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10.1146/annurev.genet.38.072902.091600
Annu. Rev. Genet. 2004. 38:1–35
doi: 10.1146/annurev.genet.38.072902.091600
c 2004 by Annual Reviews. All rights reserved
Copyright First published online as a Review in Advance on May 25, 2004
MOBILE GROUP II INTRONS
Alan M. Lambowitz1 and Steven Zimmerly2
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1
Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry,
and Section of Molecular Genetics and Microbiology, School of Biological Sciences,
University of Texas at Austin, Austin, Texas 78712; email: [email protected]
2
Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4
Canada; email: [email protected]
Key Words intron evolution, retrotransposon, reverse transcriptase, ribozyme,
RNA splicing
■ Abstract Mobile group II introns, found in bacterial and organellar genomes,
are both catalytic RNAs and retrotransposable elements. They use an extraordinary
mobility mechanism in which the excised intron RNA reverse splices directly into a
DNA target site and is then reverse transcribed by the intron-encoded protein. After
DNA insertion, the introns remove themselves by protein-assisted, autocatalytic RNA
splicing, thereby minimizing host damage. Here we discuss the experimental basis
for our current understanding of group II intron mobility mechanisms, beginning with
genetic observations in yeast mitochondria, and culminating with a detailed understanding of molecular mechanisms shared by organellar and bacterial group II introns.
We also discuss recently discovered links between group II intron mobility and DNA
replication, new insights into group II intron evolution arising from bacterial genome
sequencing, and the evolutionary relationship between group II introns and both eukaryotic spliceosomal introns and non-LTR-retrotransposons. Finally, we describe the
development of mobile group II introns into gene-targeting vectors, “targetrons,” which
have programmable target specificity.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
DISTRIBUTION OF GROUP II INTRONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
GROUP II INTRON SPLICING MECHANISM AND
RNA STRUCTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
PROTEIN-ASSISTED SPLICING OF GROUP II INTRONS . . . . . . . . . . . . . . . . . . . 5
GROUP II INTRON-ENCODED PROTEINS: DOMAINS
AND BIOCHEMICAL ACTIVITIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
GROUP II INTRON-ENCODED PROTEINS: LINEAGES
AND VARIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
DEGENERATE GROUP II INTRONS, TRANS-SPLICING,
AND TWINTRONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
RETROHOMING OF YEAST mtDNA GROUP II INTRONS . . . . . . . . . . . . . . . . . . 10
RETROHOMING OF THE L. LACTIS Ll.LtrB INTRON . . . . . . . . . . . . . . . . . . . . . . 14
0066-4197/04/1215-0001$14.00
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ALTERNATE MOBILITY MECHANISMS USED BY En−
GROUP II INTRONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GROUP II INTRON RETROTRANSPOSITION TO ECTOPIC SITES . . . . . . . . . . .
GROUP II INTRON RETROTRANSPOSITION SUPPORTED
BY AN INTRON-ENCODED PROTEIN IN TRANS? . . . . . . . . . . . . . . . . . . . . . . . .
MECHANISM OF SECOND-STRAND SYNTHESIS
AND CONNECTIONS TO DNA REPLICATION . . . . . . . . . . . . . . . . . . . . . . . . . . .
SYNTHESIS OF THE INTRON-ENCODED PROTEIN . . . . . . . . . . . . . . . . . . . . . . .
BINDING OF THE INTRON-ENCODED PROTEIN TO THE
INTRON RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DNA TARGET SITE RECOGNITION BY GROUP II INTRON RNPs . . . . . . . . . . .
REGULATION OF GROUP II INTRON MOBILITY . . . . . . . . . . . . . . . . . . . . . . . . .
EVOLUTION OF MOBILE GROUP II INTRONS . . . . . . . . . . . . . . . . . . . . . . . . . . .
EVOLUTION OF THE INTRON-ENCODED PROTEIN
AND PROTEIN-ASSISTED SPLICING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
EVOLUTIONARY RELATIONSHIP OF GROUP II INTRONS
TO SPLICEOSOMAL INTRONS AND
NON-LTR-RETROTRANSPOSONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GROUP II INTRONS AS GENE-TARGETING VECTORS . . . . . . . . . . . . . . . . . . . .
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16
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20
23
24
24
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INTRODUCTION
Mobile group II introns are retroelements consisting of a highly structured catalytic RNA and a multifunctional, intron-encoded protein (IEP), which has reverse
transcriptase (RT) activity. The intron RNA, by virtue of its ribozyme activity, intrinsically carries out RNA splicing and reverse splicing (integration) reactions,
while the IEP facilitates these reactions by stabilizing the catalytically active RNA
structure. Mobility occurs by a remarkable target DNA-primed reverse transcription (TPRT) mechanism in which the excised intron RNA reverse splices directly
into a DNA target site and is then reverse transcribed by the IEP. In some cases,
the IEP has a DNA endonuclease (En) activity that cleaves the opposite strand to
generate the primer for TPRT, but recent studies show that nascent strands at DNA
replication forks can also serve as primers. By using these mechanisms, group II
introns “retrohome” at frequencies approaching 100% into specific DNA target
sites, typically the unoccupied site in an intronless allele, and “retrotranspose”
at low frequencies into ectopic sites that resemble the normal homing site. Mobile group II introns are also of interest because they are the putative ancestors of
spliceosomal introns and non-LTR retrotransposons in higher organisms, elements
that together comprise more than 45% of the human genome.
DISTRIBUTION OF GROUP II INTRONS
Group II introns were discovered in the mitochondrial (mt) and chloroplast (cp)
genomes of lower eukaryotes and higher plants, where they interrupt conserved
genes. About a third of organellar group II introns encode an open reading frame
(ORF) with homology to RTs (68, 109). Among the first identified and studied were
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the yeast mtDNA introns coxI-I1 and -I2 (also known as aI1 and aI2, respectively),
which are found at two sites in the cytochrome oxidase subunit I gene (6). These
and other examples of closely related group II introns found at different loci,
along with their RT-related ORFs, suggested that group II introns might be mobile
elements (50).
The first bacterial group II introns were discovered about ten years ago by PCR
screens (34). Only recently, however, has genome sequencing revealed that group II
introns are surprisingly common in both gram-negative and gram-positive bacteria
(23, 111). About a quarter of sequenced bacterial genomes contain a group II
intron, with up to two dozen present in a given organism (21, 111). By comparison,
group II introns are rare in archaea, being found in only 2 closely related species
out of 17 whose genomes have been sequenced (91). Unlike organellar group
II introns, almost all the bacterial introns encode an RT-related ORF or ORF
remnant. Additionally, most bacterial group II introns are inserted either between
genes or within mobile DNAs, such as IS elements or plasmids, which may aid
their dissemination (21, 43). The well-studied Lactococcus lactis Ll.LtrB intron,
for example, was discovered in a relaxase gene (ltrB) in a conjugative element,
where its splicing is required for conjugation (72, 100).
Group II introns have not been found in the streamlined mtDNAs of metazoan animals, possibly because of intron loss, nor in eukaryotic nuclear genes,
where their presumed descendants, spliceosomal introns, predominate. However,
the nuclear genome of the higher plant Arabidopsis thaliana contains an integrated,
presumably nonfunctional mtDNA sequence that includes group II introns (57).
Nuclear integration of mtDNA fragments occurs frequently and is one process by
which group II introns may have been transferred to the nucleus before evolving
into spliceosomal introns (7).
GROUP II INTRON SPLICING MECHANISM
AND RNA STRUCTURE
Like spliceosomal introns, group II introns splice via two sequential transesterification reactions that yield ligated exons and an excised intron lariat with a 2 -5
phosphodiester bond (84, 97, 113) (Figure 1A). In the case of group II introns,
however, the splicing reactions are catalyzed by the intron RNA itself. To accomplish this, the RNA folds into conserved secondary and tertiary structures,
which form an active site containing catalytically essential Mg2+ ions (67, 89).
The conserved secondary structure consists of six double-helical domains (DIDVI) radiating from a central wheel, with three major subgroups (IIA, IIB, and
IIC) and further subdivisions defined by specific features (33, 69, 111) (Figure
1B). DV interacts with DI to form the minimal catalytic core; DVI contains the
branch-point nucleotide residue, usually a bulged A; and DII and DIII contribute
to RNA folding and catalytic efficiency (32). DIV, which encodes the intron ORF,
is dispensable for ribozyme activity. NMR and X-ray crystal structures have been
determined for segments DV and DV + DVI (102, 119), and over a dozen tertiary
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Figure 1 Group II intron RNA splicing mechanism and secondary structure. A. Splicing occurs via two sequential transesterification reactions. In the first, nucleophilic
attack at the 5 -splice site by the 2 OH of a bulged A-residue in DVI results in cleavage of the 5 -splice site coupled to formation of lariat intermediate. In the second,
nucleophilic attack at the 3 -splice site by the 3 OH of the cleaved 5 exon results in
exon ligation and release of the intron lariat. B. The conserved secondary structure
consists of six double-helical domains (DI-DVI) emanating from a central wheel, with
subdomains indicated by lower-case letters (e.g., DIVa). The ORF is encoded within
DIV (dotted loop), and DIVa is the high-affinity binding site for the IEP. Greek letters
indicate sequences involved in tertiary interactions. EBS and IBS refer to exon- and
intron-binding sites, respectively. Some key differences between subgroup IIA, IIB,
and IIC introns are indicated within dashed boxes, but additional smaller differences
are not shown (see References 69, 109 for detailed discussion of differences between
group II intron subclasses).
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interactions between different RNA domains have been identified (IBS1-3, α-λ),
enabling structural modeling of the active site (14, 82, 107). An evolutionary relationship between group II and spliceosomal introns is supported by their similar
splicing mechanisms and by the finding that several group II intron domains, including DI, DIII, and DV, and segments DI-III and DV-DVI, can function in trans
to promote splicing of mutant introns lacking the domains, as expected for the
progenitors of spliceosomal snRNAs (12, 24, 45, 70, 88).
Key to the operation of group II introns are three short sequence elements
that base pair with flanking 5 - and 3 -exon sequences to help position the splice
junctions at the intron’s active site for both RNA splicing and reverse splicing
reactions (Figure 1B) (14, 67). The sequence elements EBS1 and EBS2 (exonbinding sites 1 and 2) in DI each form 5 to 6 base pairs with the 5 -exon sequences
IBS1 and IBS2 (intron-binding sites 1 and 2). In group IIA introns, the sequence
δ adjacent to EBS1 base pairs with δ , the first 1–3 nucleotides of the 3 exon,
while in group IIB introns, the 3 exon base pairs instead with EBS3, located in a
different part of DI (Figure 1B).
PROTEIN-ASSISTED SPLICING OF GROUP II INTRONS
Although some group II introns self-splice in vitro, this reaction generally requires
nonphysiological conditions, and in vivo, proteins are required to help the intron
RNA fold into a catalytically active structure (reviewed in 51, 52). In the case of
mobile group II introns, a major protein required for splicing is the IEP, which
binds specifically to the intron RNA to stabilize the active structure (“maturase”
activity) (Figure 2) (9, 64, 77, 93). As discussed below, the IEP uses parts of
the RT domain and domain X, which likely corresponds to the RT thumb, to
bind different regions of the intron RNA (17, 74, 77). All characterized maturases
are intron-specific splicing factors, but closely related maturases may have some
cross-reactivity (9, 77, 93).
In plant chloroplasts, only one group II intron, trnK-I1 in the tRNALys gene,
encodes a maturase-related protein, and this protein, denoted MatK, may contribute
to the splicing of a number of ORF-less group II introns. This more generalized
function of MatK is supported by the nature of the splicing defects resulting from
inhibition of cp protein synthesis, and by the observation that a free-standing ORF
encoding MatK is retained in the pared down cp genomes of nonphotosynthetic
plants (reviewed in 114). MatK proteins contain a well-conserved domain X, but
have degenerate RT motifs and lack the En domain (Figure 2D) (74). In higher
plants, four other maturase-related proteins, denoted nMat-1a, -1b, -2a, and -2b,
are encoded by nuclear genes, but have mt targeting sequences and may function
in splicing one or more mt group II introns, most of which do not encode ORFs
(Figure 2E,F) (73). The nuclear-encoded nMat proteins likely originated from an
organelle group II intron. Like MatK, they have a conserved domain X, but some
have deviations in the RT domain and lack or have deviations in the En domain,
suggesting loss of mobility functions.
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Figure 2 Group II intron-encoded and related proteins. Protein coding regions are
shown as rectangles, with different shadings indicating conserved regions. For proteins
encoded within introns, the 5 and 3 exons (E1 and E2, respectively) are shown as
gray boxes, and noncoding regions of the intron RNA are thick black lines. Protein
domains are: RT, with conserved sequence blocks RT-0 to -7; domain X, associated
with maturase activity; D, DNA-binding domain; and En, DNA endonuclease domain.
The locations of intron RNA domains I–VI are indicated above the Ll.LtrB intron,
with hatch marks demarcating domain boundaries. (A) L. lactis Ll.LtrB intron. (B) S.
cerevisiae mtDNA intron coxI-I1. (C) Sinorhizobium meliloti RmInt1, which encodes a
protein lacking the En domain and has only a short C-terminal extension in the position
corresponding to domain D. (D) Arabidopsis thaliana cp tRNALys intron encoding
MatK, with degenerate but recognizable features of the RT domain. (E) and (F) A.
thaliana nuclear-encoded maturase-like proteins nMat-1a and nMat-2a, respectively.
In addition to maturases, a number of host-encoded group II intron splicing
factors have been identified genetically in yeast, algae, and higher plants (reviewed
in 52, 55). Some of these proteins may function alone, whereas others may function
in conjunction with maturases or other proteins, either by stabilizing the active RNA
structure, or by acting as RNA chaperones to resolve non-native structures that
constitute “kinetic traps” in RNA folding. The host-encoded splicing factors differ
among organisms, but a common feature is that they have or had another cellular
function (e.g., peptidyl-tRNA hydrolase and CRM-domain proteins for higher
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MOBILE GROUP II INTRONS
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plant cp DNA introns; pseudouridine synthase for a trans-spliced Chlamydomonas
reinhardtii cp DNA intron) (46, 83, 85, 108). The idiosyncratic nature of these
proteins in different organisms suggests that they were recruited to function in
splicing relatively recently in evolution, after the dispersal of the introns as mobile
genetic elements, similar to the recruitment of aminoacyl-tRNA synthetases and
other host factors to function in splicing group I introns (52, 53).
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GROUP II INTRON-ENCODED PROTEINS: DOMAINS
AND BIOCHEMICAL ACTIVITIES
Most of what is known about the biochemical activities of group II IEPs has come
from studies of the yeast mt introns coxI-I1 and -I2 and the L. lactis Ll.LtrB intron.
The proteins encoded by these introns are active RTs containing four “domains”
denoted RT, X, DNA binding (D), and DNA endonuclease (En) (Figure 2A,B)
(49, 65, 74, 93, 94). The N-terminal RT domain contains conserved sequence
blocks RT-1 to -7 found in the fingers and palm of retroviral and other RTs, as
well as an upstream region, denoted RT-0, which is characteristic of RTs of nonLTR retrotransposons, such as human LINE elements (59, 126). Domain X was
identified as a site of mutations affecting maturase activity (17, 74). It is located
just downstream of the RT domain in the position corresponding to the thumb
and connection domains of retroviral RTs, and likely corresponds to a specialized
thumb in group II intron RTs. The RT and X domains function together to bind
the intron RNA both as a substrate for RNA splicing and as a template for reverse
transcription (17, 49, 65, 115).
The C-terminal D and En domains are not required for RNA splicing, but
function in intron mobility. Domain D is not highly conserved in sequence, but
contains two functionally important regions, one consisting of a cluster of basic
amino acid residues and the other containing a predicted α-helix (94). The En
domain, which carries out second-strand DNA cleavage during mobility, contains
motifs characteristic of the H-N-H DNA endonuclease family interspersed with two
pairs of conserved cysteine residues, similar to an arrangement found in phage T4
endonuclease VII (38, 94, 101). The H-N-H motif forms part of the En active site,
which in the case of the L. lactis IEP contains a single catalytically essential Mg2+
ion. The conserved cysteine motifs appear to stabilize the higher-order structure
of the domain, but unlike EndoVII, do not contain a coordinated Zn2+ ion, at least
in the purified protein (94).
The functions of the D and En domains in the yeast and lactococcal introns
were defined by analyzing C-terminal truncations (41, 103). Deletion of the En
domain abolished only second-strand DNA cleavage, while a longer truncation
deleting both D and En also abolished reverse splicing of the intron RNA into
double-stranded DNA. The truncated protein lacking D and En could still support
residual reverse splicing into single-stranded DNA substrates, implying that D is
required primarily to access the target site in double-stranded DNA. The RT and/or
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X domains may also contribute to DNA binding, as evidenced by their ability to
support some reverse splicing into single-stranded DNA. The En domain has no
cognate in other retroelements, suggesting that it was appended to a pre-existing RT
to enhance mobility by TPRT (3, 63). Nuclear non-LTR-retrotransposons contain
two other types of En domains that have been appended to their RTs for analogous
TPRT reactions (29, 76).
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GROUP II INTRON-ENCODED PROTEINS: LINEAGES
AND VARIATIONS
More than 200 ORF-containing group II introns have been sequenced, and with a
few exceptions noted below, all encode RT-related proteins. Based on phylogenetic
analysis, the IEPs can be divided into eight major lineages denoted mitochondrial,
chloroplast-like 1 and 2, and bacterial A-E (112, 126) (Figure 3). Importantly, each
lineage of IEP is associated with a distinct RNA structural subclass, implying that
the IEP was associated with the intron RNA prior to the divergence of different
group II intron lineages, with little if any subsequent exchange of IEPs (109). The
“mitochondrial” and “chloroplast” lineages include a number of bacterial group II
Figure 3 Phylogeny of group II intron ORFs and correspondence with RNA structural
classes. Phylogenetic relationships of group II intron ORFs are summarized based on
neighbor-joining analyses (109, 112). Group II intron ORFs are divided into eight
clades, named mitochondrial, chloroplast-like 1 and 2, and bacterial A–E (112, 126).
Each ORF clade is associated with a distinct RNA structural class (IIA1, IIB1, IIB2,
IIC, two other distinct IIB-like, and two distinct IIA/B hybrid classes) (109). The
branching patterns for specific introns discussed in this review are indicated, along
with group II introns of E. coli and the archaea Methanosarcina acetivorans (M.a.),
which illustrate probable horizontal transfers.
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introns (e.g., the L. lactis Ll.LtrB intron belongs to the “mitochondrial” lineage), a
situation thought to reflect that mt and cp group II introns were derived from specific bacterial lineages. Additionally, the heterogeneous phylogenetic distribution
of group II intron subclasses suggests that horizontal transfer of bacterial group
II introns is relatively common (21, 126), and indeed, cross-species transfer by
conjugation has been demonstrated (4).
About a quarter of organellar group II introns and half of bacterial group II
introns (including all members of bacterial classes C, D, and E) encode proteins
lacking the En domain. As discussed below, some of these introns are mobile, and
their IEPs may be related to ancestral RTs that lacked the En domain. However,
phylogenetic analysis suggests that the En domain was also lost multiple times in
both bacterial and organellar lineages (126).
Finally, a small subset of fungal mtDNA group II introns is distinct in encoding
proteins of the LAGLIDADG family of group I intron homing endonucleases
(110). The LAGLIDADG proteins promote homing of group I introns by cleaving
recipient alleles to initiate double-strand break repair (DSBR) recombination, and
some have also adapted to function in RNA splicing by stabilizing the active RNA
structure (2, 51, 52). It will be of great interest to determine if these proteins have
similar functions when associated with group II introns.
DEGENERATE GROUP II INTRONS, TRANS-SPLICING,
AND TWINTRONS
Group II introns exhibit several types of structural variations that provide insight
into their evolutionary potential and constraints. First, many organellar group II
introns have degenerate RNA structures. Higher plant cp and mt group II introns, for
example, frequently have deviations from the conserved RNA secondary structure,
such as mispairings in DV and DVI, and no higher plant group II intron has been
shown to self-splice (67, 83). More extreme structural degeneration is seen among
Euglena cp group II introns, some of which (also called group III introns) are as
short as 91 nts and have only a degenerate DI and DVI (13). Also, many organellar
group II introns lack an ORF or encode proteins with degenerate RT domains (e.g.,
cp MatK proteins), suggesting loss of mobility functions (74).
Remarkably, a number of organelle group II introns are discontinuous, consisting of two or more segments encoded in different parts of the genome. Transcripts
of these segments reassociate via tertiary interactions to form a functional intron,
resulting in “trans-splicing” of the associated exons (5). The nad1 gene in many
higher plants, for example, is split into four independently transcribed segments,
which are spliced in three trans-splicing reactions. In ancestral lower plants, the
nad1 gene is continuous and does not contain introns, indicating that the gene
was split after intron insertion. In Chlamydomonas reinhardtii chloroplasts, two
trans-splicing introns are found in the psaA gene, with intron 1 transcribed in
three segments and intron 2 in two segments. Mutations in about a dozen nuclear
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genes affect the C. reinhardtii trans-splicing reactions (37, 85, 92). Notably, transsplicing appears to have evolved independently multiple times in organelles (90).
The repeated distintegration of group II introns into segments that can functionally
reassociate supports the view that a similar process was involved in the evolution
of spliceosomal introns (99).
Finally, “twintrons” are a type of structural variation in which one group II
intron has inserted into another, forming a nested set of up to four introns. In
Euglena chloroplasts, twintrons are found within housekeeping genes, and they
must be spliced sequentially starting with the innermost intron, which then yields
a continuous copy of the next intron, and so on (13). By contrast, most bacterial
twintrons are found in intergenic regions, and some bacterial clusters have asymmetric organizations, with incomplete copies of some introns (23). The insertion
of one intron into another provides a safe haven for the invading intron.
RETROHOMING OF YEAST mtDNA GROUP II INTRONS
The mobility of group II introns was first demonstrated by Meunier et al. (66) for
the S. cerevisiae mtDNA intron coxI-I1 and by Skelly et al. (105) for the related
Kluyveromyces lactis coxI-I1 intron. These investigators analyzed crosses between
haploid yeast strains containing different combinations of mtDNA introns. During
crosses, mitochondria fuse, enabling recombination between mtDNAs, which then
segregate to a homoplasmic state. Both group II introns homed to the unoccupied
site in intronless alleles at high frequency, occupying ∼90% of the progeny alleles,
and in S. cerevisiae, homing was shown to be blocked both by IEP mutations and by
intron RNA mutations that inhibit splicing. The latter finding implied a significant
difference from the DSBR mechanism used for group I intron mobility, where
splicing competence of the intron is not required.
In the first detailed study of group II intron mobility, Lazowska et al. (54)
showed that both coxI-I1 and coxI-I2 are mobile independently, but in some crosses,
coxI-I1 mobility was blocked by a small number of allele-specific sequence differences in the DNA target site. By analyzing pooled progeny, they showed that
insertion of coxI-I1 into its target site is accompanied by asymmetric coconversion
of flanking exon sequences, which extended >50 bp into the 5 exon but only
<25 bp into the 3 exon. These findings and previous characterization of cDNAs
synthesized from unspliced precursor RNA in endogenous reverse transcription
reactions (49) suggested that the mobility intermediate was a reverse transcript of
unspliced precursor RNA. This cDNA was hypothesized to begin in the 3 exon and
extend through the intron into the 5 exon, enabling intron integration into the recipient allele by recombination between homologous exon sequences (Figure 4A).
The inhibition of mobility by small DNA target site differences raised the possibility of a DNA endonuclease activity that cleaves the recipient allele, and this
possibility was supported by the subsequent identification of H-N-H DNA endonuclease motifs in the C-terminal domain of the IEPs (38, 101).
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Extending these findings, Moran et al. (78) showed that coxI-I2 mobility is
inhibited by mutations in the IEP’s RT and En domains, as well as by the deletion
of intron DV, which abolished ribozyme activity without affecting the amino acid
sequence of the IEP. Unexpectedly, mutations in the RT active site that abolished
RT activity inhibited mobility only by ∼50%, whereas mutations in the En region
reduced mobility to undetectable levels. These findings suggested two different
mobility mechanisms, both of which require the En activity—an RT-dependent
mechanism involving a cDNA intermediate, and an RT-independent DSBR mechanism initiated by En cleavage of the recipient allele, analogous to group I intron
mobility. Asymmetric coconversion of exon sequences was again observed, and
as above, the simplest interpretation was that the mobility intermediate was a reverse transcript of unspliced precursor RNA. Only later did it become clear that
the coconversion pattern was actually a composite of those for different mobility
pathways operating simultaneously (see below).
Enlightenment came from biochemical studies with yeast mt RNP preparations,
in which the mobility reactions were reconstituted in vitro, enabling the dissection of individual steps (125). First, incubation of the RNPs with double-stranded
homing site DNA showed that mobility occurs by a TPRT mechanism in which the
RNPs cleave both strands of the DNA and then use the 3 end of the cleaved antisense strand as the primer for reverse transcription of an intron RNA template. The
sense strand is cleaved precisely at the exon junction, whereas the antisense strand
is cleaved at position +10 in the 3 exon. Analysis of mutants showed that the En
activity requires not only the IEP but also splicing-competent intron RNA, which
stabilizes the IEP. Because of the previous coconversion data, the initial model
continued to assume that unspliced precursor RNA was the template for reverse
transcription (Figure 4B), analogous to the TPRT mechanism first demonstrated
by Eickbush and coworkers for the insect R2 element (58). In that mechanism,
the RT uses a site-specific endonuclease activity to cleave the DNA target site,
generating a nick that is then used as a primer for reverse transcription beginning
at the 3 end of the element’s RNA.
For group II introns, the final critical revelation came from further dissection of
the DNA cleavage reaction using the newly developed biochemical methods (118,
124). These studies showed that while antisense-strand cleavage is catalyzed by
the En domain of the IEP, sense-strand cleavage is catalyzed by the intron RNA
via a reverse splicing reaction. The latter reaction inserts the intron RNA directly
into the DNA target site where it can serve as a template for reverse transcription
primed by the 3 end of the cleaved antisense strand (Figure 4C,E). While coxI-I2
carried out mainly the first step of reverse splicing in vitro, resulting in intron lariat
RNA linked to the 3 exon, coxI-I1 carried out substantial amounts of complete
reverse splicing in vitro, resulting in insertion of linear intron RNA between the
two DNA exons. These different levels of partial and complete reverse splicing
are now known to reflect that the reaction is reversible, with excision favored for
some introns, leading to a preponderance of partially reverse spliced product in
vitro (1). Reverse transcription across the 3 -splice site blocks excision and pulls
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the equilibrium toward fully reverse spliced intron, which is presumably the preferred mobility intermediate in all cases.
The finding that the intron RNA integrates into double-stranded DNA by reverse
splicing suggested that DNA target site recognition involves the EBS-IBS and δδ pairings, which are required for reverse splicing into RNA and single-stranded
DNA substrates (see Figure 1B). As discussed below, this inference was confirmed
in subsequent studies, which showed that the IEP also contributes to DNA target
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 4 Proposed and demonstrated group II intron mobility mechanisms. A. Early
proposed mechanism based on initial genetic studies of yeast mtDNA group II intron
homing. In this mechanism, the proposed mobility intermediate is a cDNA of unspliced
precursor RNA that begins just downstream of the intron in the 3 exon and continues through the intron into the 5 exon. Homologous recombination between exon
sequences in the cDNA and an intronless allele results in insertion of the intron with
coconversion of flanking exon sequences (54, 78). B. Initially proposed TPRT mechanism in which the group II intron RNP makes a double-strand break in the recipient
allele and then uses unspliced precursor RNA as a template for reverse transcription
(125). C. Major retrohoming mechanism used by the yeast mtDNA coxI-I1 and coxI-I2
introns in which the intron RNA reverse splices into the DNA target site and is reverse
transcribed by the IEP, with integration completed by DSBR-like recombination initiated by the intron cDNA invading an intron-containing allele (30, 31, 118, 124). This
mechanism leads to coconversion of 5 - but not 3 -exon sequences. D. RT-independent
homing mechanism used by yeast mtDNA group II introns, in which group II intron
RNPs cleave the recipient allele to initiate DSBR, analogous to group I intron homing
(30, 31). This mechanism results in coconversion of both 5 - and 3 -exon sequences.
E. Major retrohoming mechanism used by the L. lactis Ll.LtrB intron and for some
homing events by the yeast mtDNA introns. The intron RNA reverse splices into the
DNA target site and is used as a template to synthesize a full-length cDNA, which is
integrated by DNA replication and repair enzymes (16, 30). F. and G. Mechanisms
used by the L. lactis Ll.LtrB intron for retrohoming in the absence of second-strand
cleavage. The intron RNA reverse splices into double-stranded DNA and uses a nascent
leading or lagging strand at a DNA replication fork as the primer for reverse transcription (122). Variations of these mechanisms may be used by naturally occurring group
II introns that encode proteins lacking the C-terminal En activity. H. Mechanism in
which the intron RNA reverse splices into single-stranded DNA at a replication fork
and uses a nascent lagging strand DNA as a primer for reverse transcription (44, 122).
This mechanism has been hypothesized for retrotransposition of the Ll.LtrB intron to
ectopic sites in L. lactis and also may be used by naturally occurring group II introns,
whose IEPs lack En activity. I. Alternate proposed mechanism for En− mobility, in
which the intron RNA reverse splices into the DNA target site and then uses a nick
in the opposite strand as a primer for reverse transcription (95). Models A and B are
shown in brackets to indicate that they have been superseded by other mechanisms
shown in the figure.
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recognition and helps promote local DNA unwinding, enabling the intron RNA to
base pair to the target DNA. By using the same base-pairing interactions with the
5 and 3 exons for both RNA splicing and DNA target site recognition, the intron
ensures that it will insert only at target sites from which it can subsequently splice,
thereby minimizing host damage.
Following the biochemical studies, genetic analysis of large numbers of individual mobility events for wild-type coxI-I1 and -I2 indicated that after reverse
splicing of the intron RNA into the DNA target site, mobility can be completed
by at least three different mechanisms (30, 31). In the major pathway (∼60% of
the events), intron insertion is accompanied by coconversion of 5 - but not 3 exon sequences. These events are thought to occur by reverse splicing of the intron
RNA into the DNA target site, followed by synthesis of a complete or partial intron
cDNA, which then invades an intron-containing allele to initiate DSBR-like recombination (Figure 4C). Completion of that process leads to variable coconversion of
5 - but not 3 -exon sequences. In the second pathway (∼40% of the events), intron
insertion is accompanied by coconversion of both 5 - and 3 -exon sequences, presumably through DSBR, initiated by RNP cleavage of the target site (Figure 4D).
As expected, this pathway remains active in RT-deficient mutants. Finally, a small
proportion of mobility events for the wild-type introns occurs without any coconversion of exon sequences, presumably via synthesis of a full-length intron cDNA,
which is integrated by DNA repair, similar to the major pathway first demonstrated
for the L. lactis Ll.LtrB intron (see below; Figure 4E). Interestingly, the proportion
of mobility events occurring by the third pathway was increased to 43% by certain
DNA target site mutations (30). Although the use of a precursor RNA template
is not needed to explain the coconversion data, it remains a possibility for some
mobility events, particularly in light of biochemical studies showing that the RT is
bound to precursor RNA in a position to initiate cDNA synthesis in the 3 exon (115,
127). The ability to complete mobility by using different cellular recombination
and repair activities makes group II introns adaptable to different host organisms.
RETROHOMING OF THE L. LACTIS Ll.LtrB INTRON
The discovery of group II introns in bacteria suggested that tractable bacterial
systems might be developed for detailed genetic and biochemical analysis (34).
However, most bacterial introns proved immobile and refractory to high-level expression in Escherichia coli. An exception was the Lactococcus lactis Ll.LtrB
intron (71, 100). Matsuura et al. (65) developed an efficient E. coli expression
system for Ll.LtrB and showed that the IEP, denoted LtrA, has RT, maturase,
and En activities. Further, RNPs consisting of the IEP and lariat RNA could reverse splice into double-stranded DNA target sites and carry out TPRT of the
inserted intron RNA. A minor difference from the yeast mt introns was that the
IEP cleaved the bottom strand at position +9 rather than +10. It was also shown
that a drug-resistance marker could be inserted into the DIV loop without impeding
the mobility reactions, demonstrating how the introns could be used as vectors.
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Next, Cousineau et al. (16) used the genetically marked introns to develop
plasmid-based genetic assays for intron homing in E. coli and L. lactis. In a key
experiment, a group I intron introduced along with a kanR marker in DIV was
absent after intron homing, proving that mobility occurs via an RNA intermediate,
from which the group I intron could splice. Retrohoming of Ll.LtrB in both E. coli
and L. lactis was found to be RecA-independent and to occur without coconversion
of flanking exon sequences (16, 71). Both properties differ from the major pathway
used by the yeast mt introns and suggest that retrohoming of Ll.LtrB occurs predominantly via the synthesis of a full-length cDNA of the reverse spliced intron,
which is then integrated by DNA repair independent of homologous recombination
(Figure 4E).
ALTERNATE MOBILITY MECHANISMS USED
BY En− GROUP II INTRONS
As mentioned previously, many bacterial group II introns encode proteins lacking
the En domain. Thus far, the best studied is Sinorhizobium meliloti RmInt1, a
bacterial class D intron found originally in an IS element (Figure 2C). RmInt1
retrohomes efficiently, inserting into 20% to 48% of plasmid-borne target sites
introduced into intron-containing strains (60). The RmInt1 IEP has RT activity,
and its RNPs can reverse splice into double- or single-stranded DNA substrates, but
cannot carry out site-specific second-strand cleavage, thus requiring an alternate
mechanism to prime reverse transcription (80).
Insight into priming mechanisms for En− introns was obtained by analyzing
mobility of the Ll.LtrB intron in E. coli, using conditions in which second-strand
cleavage was blocked by mutations in either the IEP or DNA target site (122). Such
mutations decreased but did not eliminate mobility, and the residual mobility then
showed a pronounced replication frequency dependence and strand bias, suggesting that a nascent leading strand at a DNA replication fork was used to prime reverse
transcription (Figure 4F). Residual mobility also occurred at lower frequency in the
opposite orientation, with characteristics suggesting that nascent lagging strands
could be used as primers to some extent (Figure 4G). The preference for leading
strand primers may reflect that after reverse splicing into double-stranded DNA,
the intron RNP is positioned to use a leading strand primer directly, while the
use of a lagging strand primer requires the potentially disruptive passage of the
replication fork through the region containing the inserted intron RNP. Notably,
En− retrohoming of the Ll.LtrB intron is dependent upon the RT activity of the
IEP, suggesting that the host DNA polymerase does not simply copy through the
inserted intron RNA and that the nascent DNA strand is used instead to prime
reverse transcription by the IEP. The situation may be analogous to the mode of
action of lesion bypass DNA polymerases, where the host replicative polymerase
disengages and then resumes after the lesion is repaired (87).
Similar mechanisms may be used by naturally occurring group II introns that
lack the En domain. Ichiyanagi et al. (44) noted that most En− introns are found
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on the lagging template strand, the orientation opposite that for retrohoming of
En− mutants of the Ll.LtrB intron. This opposite strand bias may reflect that the En−
introns retrohome by reverse splicing into single-stranded DNA after the passage
of the replication fork, enabling facile use of lagging strand primers (Figure 4H).
En− introns that retrohome efficiently may have specific adaptations for targeting
single-stranded regions or interactions with the host replication machinery that
facilitate use of nascent DNA strands as primers. Other priming mechanisms are
also possible. Schizosaccharomyces pombe cob-I1, for example, which encodes an
IEP with an inactive En domain, has been suggested to prime reverse transcription
of the reverse spliced intron RNA by using a nonspecific opposite-strand nick
(Figure 4I) (95).
GROUP II INTRON RETROTRANSPOSITION
TO ECTOPIC SITES
In addition to retrohoming, group II introns retrotranspose at low frequency (typically 10−4 to 10−5; 25, 44) to ectopic sites that resemble the normal homing site,
providing a means of intron dispersal. Retrotransposition was first demonstrated for
group II introns in yeast and Podospora anserina mtDNAs (79, 98). In these small
genomes, duplication of the intron could be detected by PCR, but homologous
recombination rapidly deleted one copy of the intron along with the intervening
mtDNA sequences. The RmInt1 and Ll.LtrB introns expressed from plasmids were
also shown to retrotranspose to ectopic sites in their natural hosts (15, 44, 62). In
all cases, the retrotransposition sites generally had good matches for IBS1, but
poorer matches for IBS2 or for 5 - and 3 -exon sequences recognized by the IEP.
As discussed below, IEP interactions with the 5 exon are required for efficient
reverse splicing into double-stranded DNA, and interactions with the 3 exon are
required for second-strand cleavage. As expected from the lack of appropriate 3 exon sequences, retrotransposition of the Ll.LtrB intron in L. lactis does not require
the En activity of the IEP (15, 44). The initial studies of group II intron retrotransposition were interpreted in terms of an RNA-based mechanism first suggested
in general terms by Cech (10), who noted that self-splicing introns could reverse
splice into an ectopic site in RNA, leading to a recombined RNA, which is then
reverse transcribed and integrated into the genome by homologous recombination.
The finding that group II intron RNPs reverse splice into DNA homing sites
suggested an alternate “DNA target” mechanism for retrotransposition, in which
the intron RNA reverse splices directly into an ectopic DNA site and is then reverse
transcribed by the IEP (118, 124). A DNA target mechanism was supported initially
by biochemical studies with yeast coxI-I1, which showed that RNPs could reverse
splice albeit inefficiently into known DNA transposition sites in vitro (117). It was
then proven by showing that “flipping” the target sequence to the opposite strand
to prevent its transcription did not inhibit retrotransposition (25). The generality
of the mechanism was suggested by the observation that many group II intron
integration sites in bacterial genomes are located in nontranscribed intergenic
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MOBILE GROUP II INTRONS
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regions, inconsistent with an RNA target mechanism (21, 44, 61). Early experiments with the S. meliloti RmInt1 intron also suggested a DNA-target mechanism
by showing RecA-independent insertion into an ectopic site in the oxi1 gene, but
its relationship to other retrotransposition events is unclear because the oxi1 site
closely resembles the normal homing site and supports mobility at a relatively high
frequency (∼5% of the wild-type level) (62).
Ichiyanagi et al. (44) obtained further insight into group II intron retrotransposition mechanisms by a systematic study of the L. lactis Ll.LtrB intron using a powerful new genetic assay based on incorporation of a Retrotransposition Indicator
Gene (RIG) marker. This marker, analogous to those used to study retrotransposition in a variety of other systems (19), consists of a kanR gene inserted into group II
intron DIV in the reverse orientation, but interrupted by an efficiently self-splicing
group I intron in the forward orientation. During retrotransposition via an RNA
intermediate, the group I intron is spliced, reconstituting the kanR marker gene,
which is then selected after the intron has integrated into a retrotransposition site.
Analysis of a large number of events showed that retrotransposition of the Ll.LtrB
intron in L. lactis occurs into both transcribed and nontranscribed strands and
established that retrotransposition is not dependent on RecA function, a feature
obscured by selection biases in earlier experiments. In addition, most of the retrotransposition sites were found in the lagging template strand. This finding together
with the lack of 5 - and 3 -exon sequences recognized by the IEP strongly suggest
a mechanism in which the intron reverse splices into transiently single-stranded
DNA at a replication fork, followed by priming from a nascent lagging DNA
strand (43, 44, 122). In principle, retrotransposition could also occur by inaccurate
reverse splicing into double-stranded DNA, using either nascent DNA strands or
opposite-strand nicks to prime reverse transcription, with the proportion of events
occurring by different pathways varying for other introns or even for the same
intron under different conditions.
Although the DNA-target mechanism is predominant, one can ask if the RNAtarget mechanism is ever used and if not, why not? All the required steps appear to
be feasible. Group II introns reverse splice readily into RNA substrates in vitro, and
precise intron deletion from yeast mtDNA genes appears to occur by a mechanism
involving recombination between genomic DNA and a cDNA of spliced mRNA
synthesized by a group II IEP (56). A possible explanation is that for the introns
analyzed thus far, the IEP has a high affinity for DNA, thereby targeting the intron
to reverse splice preferentially into DNA sites. It remains important to look for
cases in which group II introns retrotranspose via the RNA target mechanism.
GROUP II INTRON RETROTRANSPOSITION SUPPORTED
BY AN INTRON-ENCODED PROTEIN IN TRANS?
Although most bacterial group II introns encode ORFs, four ORF-less introns were
identified recently in cyanobacteria and archaea (23, 81; L. Dai & S.Z., unpublished
data). These ORF-less introns appear to be mobile, as judged by their presence at
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multiple sites, and in each case, a closely related intron encoding an RT ORF is
also found within the genome. The latter finding raises the possibility that mobility
is promoted by the IEP in trans. The intron-insertion sites for the ORF-less introns
lack shared features except for matches for the IBS1 and IBS2 sequences recognized by base pairing of the intron RNA, suggesting reverse splicing into singlestranded DNA or RNA target sites (see above). An analogous situation may exist in
Euglena cp DNA, which contains only two protein-encoding group II or III introns,
but has a large number of ORF-less group II introns that appear to have retrotransposed by a mechanism dependent mainly on the IBS1/EBS1 pairing (13, 26, 120).
MECHANISM OF SECOND-STRAND SYNTHESIS
AND CONNECTIONS TO DNA REPLICATION
Following synthesis of the intron cDNA, both retrohoming and retrotransposition
require second-strand synthesis to complete intron integration. In the case of the
Ll.LtrB intron, the IEP has very low processive DNA-dependent DNA polymerase
activity, suggesting that second-strand synthesis may be carried out by host enzymes (J. Zhong & A.M.L., unpublished data). Consistent with this possibility, a
real-time PCR experiment using a recipient plasmid with a temperature-sensitive
replication origin showed that homing of the wild-type Ll.LtrB intron is blocked
when plasmid replication is inhibited, indicating a requirement for intron insertion
into actively replicating DNA (122). A connection between mobility and DNA
replication was also suggested by the finding that an Ll.LtrB intron with randomized EBS1/2 and δ sequences inserts preferentially at sites near the chromosome
replication origin (57% of the insertion sites found within 5% of the genome on
either side of oriC;121). Although this clustering may reflect in part the higher
copy number of origin proximal genes, which in rapidly dividing E. coli may be
greater than 4:1, it is not observed for other transposons. It could also reflect a direct interaction between group II intron RNPs and the DNA replication machinery
that leads to intron insertion soon after initiation of DNA synthesis at OriC.
SYNTHESIS OF THE INTRON-ENCODED PROTEIN
Studies with the yeast mtDNA and lactococcal Ll.LtrB introns have provided
insight into individual steps in intron mobility. The IEP, which is required for RNA
splicing, must be translated initially from unspliced precursor RNA. In Ll.LtrB and
other bacterial group II introns, the ribosome-binding site and initiation codon of
the intron ORF are located in or near the DIVa stem-loop structure, which is a highaffinity binding site for the IEP (115). Experiments with Ll.LtrB showed that the
IEP functions most efficiently when expressed in cis from the same plasmid as the
intron RNA (17, 123), and that its binding to DIVa down-regulates translation by
sequestering the ribosome-binding site (104). The latter prevents the accumulation
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of excess IEP and also halts ribosome entry into the intron, which might otherwise
impede RNA splicing. In L. lactis, the Ll.LtrB intron may also use an internal
promoter in DI to independently express LtrA (123), but there is no evidence
that such an internal promoter is used in E. coli where Ll.LtrB stills functions
efficiently. Many organellar group II introns, including yeast coxI-I1 and -I2, differ
from bacterial introns in that the ORF extends upstream from DIV and is translated
in frame with the upstream exon, yielding a precursor protein that is cleaved to
generate the active IEP (67). In these cases, the splicing of the intron prevents
further translation, again feedback regulating the IEP (9). The upstream extension
of the ORF in organellar group II introns is thought to be an evolutionary adaptation
that permits more efficient translation initiation or regulation (51).
BINDING OF THE INTRON-ENCODED PROTEIN
TO THE INTRON RNA
The interaction between the IEP and intron RNA is critical for all steps of intron
splicing and mobility. The RNA splicing activity of the IEP was first demonstrated
genetically for the yeast mtDNA coxI-I1 and -I2 introns (9, 77) and subsequently
both genetically and biochemically for L. lactis Ll.LtrB intron (65). The biochemical studies with the Ll.LtrB intron showed that the purified LtrA protein
binds tightly and specifically to the Ll.LtrB intron but not to noncognate group
II introns, and is by itself sufficient to promote RNA splicing at physiological
Mg2+concentrations in vitro (93). The LtrA protein binds to the intron RNA as a
dimer, the same quaternary structure found for other RTs (93).
Further studies with Ll.LtrB suggested a model in which the IEP binds first
to the high-affinity binding site in DIVa, where it also autoregulates translation
(see above), and then makes secondary contacts with conserved catalytic core
regions, potentially including DI, DII, and DVI, to fold the intron RNA into the
active structure (64, 115). Analysis of mutants suggests that the N terminus of the
RT domain interacts with DIVa, whereas other regions of the RT and X domains
interact with the catalytic core (17). There is some indication that after the initial
binding to DIVa, the interaction of the IEP with the catalytic core occurs by tertiarystructure capture rather than by tertiary-structure induction (82). The yeast coxI-I2
intron uses a similar mechanism in which DIVa is required for stable binding of
the IEP and additional contacts with the catalytic core promote RNA splicing (42).
The high-affinity binding to DIVa accounts at least in part for the intron specificity
of maturases and may also serve to anchor or guide the interaction of the IEP with
catalytic core regions.
Although DIVa makes a strong contribution to binding, residual splicing of
Ll.LtrB and yeast coxI-I2 can occur in the absence of DIVa by direct binding of
the IEP to the catalytic core both in vitro (64, 115) and in vivo (17, 42). The level of
residual splicing in vivo in the absence of DIVa is considerably different for Ll.LtrB
and coxI-I2 (10% and 70%, respectively), possibly reflecting different relative
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affinities of the IEP for different binding sites. However, for both introns, deletion
of DIVa has a much stronger effect on intron mobility (>105-fold inhibition; 20,
42). The latter could reflect the fact that binding to DIVa is particularly critical
for positioning the RT to initiate reverse transcription (115). The finding that the
IEP can interact directly with the conserved regions of the intron, independent of
DIVa, suggests how maturases might evolve from intron-specific to general group
II intron splicing factors, as may have occurred for the plant MatK and nMat
proteins (64).
The use of a high-affinity binding site outside the catalytic core differs from
the mode of action of well-studied group I intron splicing factors, which bind
directly to the catalytic core to stabilize the active RNA structure (reviewed in 52).
A possible rationale is that high-affinity binding to DIVa anchors one region of the
protein to the intron RNA, while leaving other regions relatively flexible to engage
in different interactions during the multiple steps in RNA splicing and intron
mobility. In the latter process, the IEP sequentially recognizes the DNA target site,
promotes reverse splicing, cleaves the second strand, and initiates TPRT, all while
remaining associated in some way with the intron RNA (64).
DNA TARGET SITE RECOGNITION BY GROUP II
INTRON RNPs
Group II intron RNPs recognize DNA target sequences by using both the IEP and
base pairing of the intron RNA. Figure 5A–D compare the DNA target sites for four
group II introns analyzed thus far. In all four cases, the intron RNA base pairs to the
5 - and 3 -exon sequences via the previously discussed EBS2/IBS2, EBS1/IBS1
and δ-δ (group IIA), or EBS3/IBS3 (group IIB) interactions, while the IEP recognizes additional upstream and downstream sequences (41, 47, 75, 103, 117).
In each case, the importance of the base-pairing interactions was demonstrated
by showing that mutations in the DNA target site could be rescued by compensatory mutations in the intron RNA. Notably, analysis of reverse splicing into RNA
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 5 DNA target site recognition by mobile group II introns. Target sites are
shown for the following group II introns: A. L. lactis Ll.LtrB (40, 75, 86); B. S. cerevisiae
coxI-I1 (117); C. S. cerevisiae coxI-I2 (41); D. S. meliloti RmInt1 (47); and E. bacterial
class C introns (21, 39). Group II intron RNPs use both the IEP and base pairing
of the intron RNA to recognize specific sequences in the DNA target site. The most
critical positions recognized by the IEP in the 5 and 3 exons are indicated by shaded
squares and ovals, respectively, based on original references, where they are defined
by somewhat different criteria. The IBS and δ sequence regions recognized by base
pairing of the intron RNA’s EBS and δ sequences are also shaded. Bacterial class
C introns appear to recognize the inverted repeat of a rho-independent transcription
terminator followed by an IBS1 sequence that base pairs with the intron RNA.
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substrates showed that single-base mismatches affect the kcat for reverse splicing
as well as Kd, and thus have a much stronger inhibitory effect than is expected from
decreased binding affinity alone (116). This feature likely contributes to the very
high target specificity for intron insertion. The DNA target sequences recognized
by the IEP differ even for closely related introns, such as coxI-I1 and -I2, suggesting
that the IEP can evolve readily to recognize different target sites, a feature that
may help group II introns to establish themselves at new locations. Further, for all
of the introns, relatively few positions are recognized by the IEP, implying that
most of the specificity for DNA target site recognition comes from base pairing of
the intron RNA.
The yeast mtDNA and lactococcal introns, whose IEPs contain C-terminal D
and En domains, recognize relatively long DNA target sites (30–35 bp). For all
three introns, mutations in key nucleotide residues recognized by the IEP in the
distal 5 -exon region inhibit both reverse splicing and second-strand cleavage,
whereas 3 -exon mutations inhibit only second-strand cleavage. In the case of
RmInt1, whose IEP lacks the En domain as well as all or part of domain D, DNA
target site recognition by the IEP appears more limited, with mutations at only two
positions (−15 and +4) strongly inhibiting mobility (47). Since RmInt1 does not
carry out second-strand cleavage, IEP recognition of the 3 -exon must be required
either for the initial DNA target site recognition or for reverse splicing, a difference
from the yeast and lactococcal introns.
Thus far, detailed biochemical analysis of DNA target site recognition has been
done only for the Ll.LtrB intron, where an efficient E. coli expression system
makes it possible to obtain large amounts of reconstituted RNPs for biochemical
studies (93). Kinetic analysis showed that the RNPs first bind DNA nonspecifically
and then search for their target site sequence, presumably by facilitated-diffusion
mechanisms analogous to those used by site-specific DNA-binding proteins (1).
The initial recognition event appears to involve major groove interactions between the IEP and key bases in the distal 5 -exon region, including T-23, G-21,
and A-20 on the same strand into which the intron reverse splices (Figure 5A)
(103). These base interactions bolstered by phosphate-backbone contacts trigger
local DNA unwinding, enabling the intron RNA to base pair to the IBS and δ sequences for reverse splicing. Mutagenesis experiments indicate some preferences
for specific bases from positions −17 to −13, where the IEP crosses the minor
groove, but it is not clear if this is due to direct interactions with bases or indirect
readout of DNA structure (86). Importantly, mutations at all critical bases in the
distal 5 -exon region strongly inhibit reverse splicing into double-stranded DNA
but have little if any effect on reverse splicing into otherwise identical singlestranded DNA targets, implying that their recognition is required mainly for DNA
unwinding (122). Second-strand cleavage occurs after a lag and is dependent on
the same protein and base-pairing interactions that position the RNP for reverse
splicing (although reverse splicing per se is not required; 1), as well as a small
number of additional IEP interactions with the 3 exon, the most critical being
recognition of T + 5 (Figure 5A) (75, 103). The latter lies in the region of the
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MOBILE GROUP II INTRONS
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DNA target site that becomes single-stranded after DNA unwinding, suggesting a mechanism for temporally separating reverse splicing and second-strand
cleavage.
The small number of bases recognized by the IEP in the distal 5 -exon region raises the question of how RNPs recognize sites that can base pair with
the intron RNA without inefficiently unwinding a large number of noncognate
sites. The answer seems to be that DNA unwinding requires concerted interaction of the IEP and base-pairing of the intron RNA, which may drive the process to completion. Thus, RNPs reconstituted with wild-type IEP and an intron
RNA, whose EBS and δ sequences were modified to prevent base pairing with the
DNA target site, did not induce DNA unwinding assayed by KMnO4 modification (103). The concerted RNA interaction could involve progressive sampling of
base pairs starting from one end, or possibly triplex formation followed by base
pairing.
Bacterial class C introns, whose IEPs lack an En domain, have a highly distinct
target specificity. These introns insert downstream of palindromic rho-independent
transcription terminators at sites having potential IBS1 but not IBS2 sequences
(Figure 5E). Moreover, bacterial class C introns are often found inserted at multiple
target sites that have little sequence similarity, but share the palindromic terminator
motif, suggesting IEP recognition of higher-order DNA structure (21, 39). Indeed,
the formation of a DNA hairpin structure would be favored in single-stranded
regions and may be a key factor in targeting these En− introns to the lagging
template strand at DNA replication forks, where they can use a nascent lagging
strand to prime reverse transcription (see above).
REGULATION OF GROUP II INTRON MOBILITY
Mobile elements and their hosts use diverse mechanisms to regulate transposition
and thus minimize host damage (8). For group II introns, the primary mechanisms are site-specific insertion and removal by RNA splicing. In addition, many
bacterial group II introns insert into benign sites, such as downstream of transcription terminators or within mobile elements, pathogenicity islands, or other group II
introns (21, 23). In a number of cases, these target sites correspond to conserved sequences, e.g., conserved regions of transposases or the RT domain of other group II
introns (23, 106), maximizing the chances that unoccupied sites will be available.
More direct regulation is also possible. Some E. coli strains, for example, contain
structurally intact group II introns but also have unfilled homing sites, suggesting
that mobility is rare, and attempts to demonstrate mobility of E. coli introns have
failed (22). The mobility of group II introns in E. coli is impeded by endogenous ribonucleases, which nick the intron RNA (40). However, this factor alone
seems insufficient to account for the immobility of E. coli introns, implying additional mechanisms that suppress group II intron mobility or limit it to specific
environmental conditions.
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EVOLUTION OF MOBILE GROUP II INTRONS
The phylogenetic distribution of mobile group II introns suggests that they evolved
in bacteria and were then transferred to eukaryotes, possibly via bacterial endosymbionts that gave rise to organelles (34, 111, 115, 126). An origin in archaea seems
less likely since the few group II introns found in archaea can be accounted for
by horizontal transfer from bacteria (91). The inference from phylogenetic analysis that the IEP was associated with the intron RNA prior to the divergence of
different group II intron lineages (see above) led to the “retroelement ancestor
hypothesis.” According to this hypothesis, all extant group II introns descended
from RT-encoding group II introns in bacteria, with two lineages becoming associated with organelles, and ORF loss occurring in many organellar and a few
bacterial group II introns (109). This hypothesis is supported by the finding of
ORF remnants in many, but not all, ORF-less introns (23, 109).
The “retroelement ancestor hypothesis” does not directly address how mobile
group II introns originated. One possibility is that they arose from a retroelement
that developed self-splicing activity to minimize deleterious effects of its transposition on the host (18). However, this scenario does not provide a compelling
rationale for the evolution of a complex ribozyme structure, since the same RNA
splicing reactions are readily carried out by protein enzymes. An alternate hypothesis is that mobile group II introns were created by the insertion of a retroelement
or RT into a pre-existing group II ribozyme, analogous to the evolution of mobile
group I introns by the invasion of ORFs encoding DNA homing endonucleases
(51). In this scenario, the group II ribozyme originated before its association with
the RT and may have functioned as a mobile element by reverse splicing into
RNA or DNA sites. The insertion of an RT into the intron would then have resulted in a more efficient genomic parasite [see Discussion in (115)]. An interesting possibility is that group II intron ribozymes evolved initially in thermophiles
or halophiles, providing conditions in which the intron RNA might be catalytically efficient. Acquisition of the ORF might then have enabled the introns to
break out into other bacterial species. This hypothesis predicts that self-sufficient
ORF-less group II introns may still exist in organisms that live under extreme
conditions.
EVOLUTION OF THE INTRON-ENCODED PROTEIN
AND PROTEIN-ASSISTED SPLICING
In either scenario for the origin of mobile introns, the RT functioned initially in
intron mobility and then adapted to function in RNA splicing by virtue of its
specific interaction with the intron RNA. The ancestral RT likely lacked the D and
En domains and may have promoted mobility by one of the mechanisms discussed
previously for En− group II introns, e.g., reverse splicing into transiently singlestranded DNA followed by use of a nascent DNA strand at a replication fork to
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prime reverse transcription (Figure 4H). Acquisition of the D and En domains
would have allowed efficient mobility by reverse splicing into double-stranded
DNA without the need for DNA replication to generate a primer (Figure 4C–E).
Group II introns that insert within genes are under selective pressure to retain
splicing activity and to limit mobility in order to minimize host damage. Both
requirements are satisfied by the replacement of the IEP by cellular splicing factors,
as observed for organellar group II introns. Further, loss of mobility functions is
expected for elements that have saturated available target sites, because there is
less opportunity to select functional variants (36). The saturation of available target
sites and fewer opportunities for horizontal transfer may explain why degenerate
group II introns are much more prevalent in small organelle genomes than in
bacteria (21).
The different proteins recruited to promote splicing in different hosts may
in turn have dictated distinct patterns of RNA structural degeneration. Thus,
fungal mtDNA group II introns, which retain the canonical structure but selfsplice inefficiently, may rely on splicing factors that compensate only for limited
RNA structural defects, whereas higher plant cp and mt group II introns, which
have greater structural deviations, may rely on a larger number of more globally acting proteins, perhaps acting together in a complex (83, 92, 108). Similarly, the wholesale structural degeneration of Euglena cp group II introns may
reflect the recruitment of proteins that supply the missing functions or a stochastic event that enabled some group II intron domains to function efficiently in
trans.
EVOLUTIONARY RELATIONSHIP OF GROUP II
INTRONS TO SPLICEOSOMAL INTRONS AND
NON-LTR-RETROTRANSPOSONS
Group II introns are the proposed ancestors of both nuclear spliceosomal introns
and nuclear non-LTR-retrotransposons (11, 27, 28, 99, 125). The key steps postulated to have occurred in the conversion of group II introns into spliceosomal
introns—degeneration of internal RNA structure, dependence on a common splicing apparatus, and the use of trans-acting RNAs—are all exemplified by the properties of group II introns in different organisms. The involvement of snRNAs in
the splicing of nuclear introns seems a particularly compelling argument for such
a scenario, since RNA splicing reactions per se can be catalyzed simply by protein
enzymes, as for eukaryotic tRNA introns.
In eukaryotes, the nuclear envelope, which separates transcription from at least
the bulk of translation, would constitute a barrier for both splicing and mobility
of group II introns, since the IEP can no longer bind the intron RNA immediately
after transcription. This separation may favor the substitution of host-encoded
proteins that could function more efficiently in trans, leading to the evolution of a
common splicing apparatus. It is possible that early “spliceosomal” introns retained
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mobility by interacting with the RTs in trans, as may be the case for some presentday ORF-less introns (see above). However, as the number of introns grew, mobility
would be increasingly detrimental to the host, favoring the replacement of the
RT with other cellular splicing factors. Group II introns that had not inserted
within genes would be under no selective pressure to retain splicing, enabling them
to evolve into non-LTR-retrotransposons. Although it is uncertain whether any
evolutionary scenario can be proven, additional clues may be obtained by searching
the genomes of primitive eukaryotes for remnants of group II introns or primitive
snRNAs.
GROUP II INTRONS AS GENE-TARGETING VECTORS
Because group II introns recognize their DNA target sites mainly by base pairing
of the intron RNA, they can be targeted to insert into different DNA sites simply
by modifying the intron RNA (31, 40, 41). This feature, combined with their very
high insertion frequency and specificity, has made it possible to use mobile group II
introns as programmable gene-targeting vectors, dubbed “targetrons.” A targetron
derived from the L. lactis Ll.LtrB intron has been used for efficient targeted gene
disruption in both gram-negative and gram-positive bacteria (35, 48, 86, 121). Additionally, group II introns can be used for the site-specific chromosomal insertion
of cargo genes cloned in DIV (e.g., 35) and to introduce targeted double-strand
breaks, which stimulate homologous recombination with a cotransformed DNA
fragment, enabling the introduction of point mutations (48).
In bacteria, retargeted group II introns are generally expressed from a donor
plasmid. The donor plasmid pACD3, shown in Figure 6, expresses an Ll.LtrBORF intron and short flanking exons, with the IEP synthesized from a position
just downstream of the 3 exon (121). The IEP expressed from this position still
promotes efficient splicing and mobility, but after insertion at a new location, the
ORF intron is unable to splice significantly in the absence of the IEP, yielding
a gene disruption. Since the ORF intron contains multiple stop codons in all
reading frames, suitably placed disruptions are likely to ablate gene function. An
intron targeted to the antisense strand inserts in the orientation opposite target
gene transcription and cannot be spliced, yielding an unconditional disruption.
By contrast, an intron targeted to the sense strand inserts in the same orientation
as target gene transcription, and a ORF intron inserted in this orientation may
give a conditional disruption, if its splicing is linked to the expression of the
IEP from a separate construct (Figure 6) (35, 48). Group II intron retargeting is
now done routinely by using a computer program that scans the desired target
sequence for the best matches to the positions recognized by the IEP and then
designs primers for modifying the intron’s EBS and δ sequences to insert into
those sites (86). The positions recognized by the IEP are sufficiently few and
flexible that the program readily identifies multiple rank-ordered target sites in any
gene.
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MOBILE GROUP II INTRONS
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Figure 6 Use of targeted group II introns (targetrons) for gene disruption. The donor
plasmid pACD3 expresses a 0.9-kb L. lactis Ll.LtrB-ORF intron and short flanking
exons, with the IEP (LtrA protein) expressed from a position just downstream of the 3
exon (121). T1 and T2 are transcription terminators. The intron is retargeted by modifying the EBS2, EBS1, and δ sequences to base pair to the IBS2, IBS1, and δ sequences
in the DNA target site. The IBS1 and 2 sequences in the donor plasmid’s 5 exon are
also modified to base pair to the intron’s retargeted EBS1 and 2 sequences for efficient
RNA splicing. Sequences modified for retargeting are boxed. An intron targeted to the
antisense strand inserts in the orientation opposite target gene transcription and thus
cannot be spliced, yielding an unconditional disruption (right). By contrast, an intron
targeted to the sense strand inserts in correct orientation to be spliced; a ORF intron
inserted in this orientation can potentially yield a conditional disruption by linking its
splicing to expression the LtrA protein in trans (35) (left).
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In E. coli, retargeted group II introns commonly insert specifically into the
desired chromosomal target site at frequencies >1% without selection (86). This
frequency is high enough to detect insertions by colony PCR. Alternatively, insertions can be detected by using a conventional or Retrotransposition-Activated
selectable Marker (RAM), patterned after previously developed RIG markers
(see above), inserted in DIV (121). One RAM marker used for gene targeting
is a small trimethoprim-resistance (TpR) gene inserted in DIV in the reverse
orientation, but interrupted by an efficiently self-splicing group I intron in the
forward orientation. The latter is excised during retrotranspositon, enabling selection of the marker after integration into a DNA target site. Even for inefficient introns, nearly 100% of the TpR colonies had the desired single disruption
(121).
In addition to targeted gene disruption, an Ll.LtrB intron containing a RAM
marker and randomized target site recognition (EBS and δ) sequences was used
to obtain disruptions at sites distributed throughout the E. coli genome, analogous
to global transposon mutagenesis. Despite clustering of insertion sites near the
chromosome replication origin, the resulting library was sufficiently complex to
contain disruptants of most if not all nonessential E. coli genes (121). Advantages
of targetrons are that each gene can be targeted individually, with multiple introns
if necessary, and that the high insertion frequencies in the absence of selection
facilitate the construction of strains having multiple disruptions or other desirable
combinations of traits.
In initial work toward developing group II intron-based gene targeting methods
in higher organisms, group II introns were designed and selected to insert into
the HIV1 provirus and the human gene encoding CCR5, an important target site
in anti-HIV therapy. The retargeted intron RNPs retained activity in human cells,
inserting into plasmid-borne HIV1 and CCR5 target sites after liposome-mediated
transfection (40). If group II introns can be adapted to function as efficiently in
gene targeting in eukaryotes as they now do in bacteria, they would have potentially
widespread applications, including the production of genetically stable disruptants
for functional genomics, and the site-specific insertion or repair of genes for gene
therapy.
ACKNOWLEDGMENTS
We thank Marlene Belfort, Georg Mohr, Philip Perlman, and Roland Saldanha for
comments on the manuscript. Work in the authors’ laboratories was supported by
NIH grants GM37949 and GM37951 to A.M.L. and CIHR, NSERC, and AHFMR
grants to S.Z. Both authors are inventors on patents and patent applications for
group II intron-based gene targeting methods assigned to the Ohio State University
and the University of Texas at Austin. They may receive a percentage of royalties
paid to the universities from licensing of those patents. In addition, A.M.L. holds
a minority equity interest in a company (InGex LLC) licensed by the universities
to commercialize group II intron gene targeting technology.
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Annual Review of Genetics
Volume 38, 2004
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CONTENTS
MOBILE GROUP II INTRONS, Alan M. Lambowitz and Steven Zimmerly
THE GENETICS OF MAIZE EVOLUTION, John Doebley
GENETIC CONTROL OF RETROVIRUS SUSCEPTIBILITY IN MAMMALIAN
CELLS, Stephen P. Goff
LIGHT SIGNAL TRANSDUCTION IN HIGHER PLANTS, Meng Chen,
Joanne Chory, and Christian Fankhauser
1
37
61
87
CHLAMYDOMONAS REINHARDTII IN THE LANDSCAPE OF PIGMENTS,
Arthur R. Grossman, Martin Lohr, and Chung Soon Im
THE GENETICS OF GEOCHEMISTRY, Laura R. Croal, Jeffrey A. Gralnick,
Davin Malasarn, and Dianne K. Newman
CLOSING MITOSIS: THE FUNCTIONS OF THE CDC14 PHOSPHATASE AND
ITS REGULATION, Frank Stegmeier and Angelika Amon
RECOMBINATION PROTEINS IN YEAST, Berit Olsen Krogh
and Lorraine S. Symington
119
175
203
233
DEVELOPMENTAL GENE AMPLIFICATION AND ORIGIN REGULATION,
John Tower
273
THE FUNCTION OF NUCLEAR ARCHITECTURE: A GENETIC APPROACH,
Angela Taddei, Florence Hediger, Frank R. Neumann,
and Susan M. Gasser
305
GENETIC MODELS IN PATHOGENESIS, Elizabeth Pradel
and Jonathan J. Ewbank
347
MELANOCYTES AND THE MICROPHTHALMIA TRANSCRIPTION FACTOR
NETWORK, Eirı́kur Steingrı́msson, Neal G. Copeland,
and Nancy A. Jenkins
EPIGENETIC REGULATION OF CELLULAR MEMORY BY THE POLYCOMB
AND TRITHORAX GROUP PROTEINS, Leonie Ringrose and Renato Paro
REPAIR AND GENETIC CONSEQUENCES OF ENDOGENOUS DNA BASE
DAMAGE IN MAMMALIAN CELLS, Deborah E. Barnes
and Tomas Lindahl
365
413
445
MITOCHONDRIA OF PROTISTS, Michael W. Gray, B. Franz Lang,
and Gertraud Burger
477
v
P1: JRX
October 18, 2004
vi
14:55
Annual Reviews
AR230-FM
CONTENTS
METAGENOMICS: GENOMIC ANALYSIS OF MICROBIAL COMMUNITIES,
Christian S. Riesenfeld, Patrick D. Schloss, and Jo Handelsman
525
GENOMIC IMPRINTING AND KINSHIP: HOW GOOD IS THE EVIDENCE?,
David Haig
553
MECHANISMS OF PATTERN FORMATION IN PLANT EMBRYOGENESIS,
Viola Willemsen and Ben Scheres
Annu. Rev. Genet. 2004.38:1-35. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF CALGARY on 02/25/05. For personal use only.
DUPLICATION AND DIVERGENCE: THE EVOLUTION OF NEW GENES
AND OLD IDEAS, John S. Taylor and Jeroen Raes
GENETIC ANALYSES FROM ANCIENT DNA, Svante Pääbo,
Hendrik Poinar, David Serre, Viviane Jaenicke-Despres, Juliane Hebler,
Nadin Rohland, Melanie Kuch, Johannes Krause, Linda Vigilant,
and Michael Hofreiter
587
615
645
PRION GENETICS: NEW RULES FOR A NEW KIND OF GENE,
Reed B. Wickner, Herman K. Edskes, Eric D. Ross, Michael M. Pierce,
Ulrich Baxa, Andreas Brachmann, and Frank Shewmaker
681
PROTEOLYSIS AS A REGULATORY MECHANISM, Michael Ehrmann and
Tim Clausen
709
MECHANISMS OF MAP KINASE SIGNALING SPECIFICITY IN
SACCHAROMYCES CEREVISIAE, Monica A. Schwartz
and Hiten D. Madhani
725
rRNA TRANSCRIPTION IN ESCHERICHIA COLI, Brian J. Paul, Wilma Ross,
Tamas Gaal, and Richard L. Gourse
749
COMPARATIVE GENOMIC STRUCTURE OF PROKARYOTES,
Stephen D. Bentley and Julian Parkhill
771
SPECIES SPECIFICITY IN POLLEN-PISTIL INTERACTIONS,
Robert Swanson, Anna F. Edlund, and Daphne Preuss
793
INTEGRATION OF ADENO-ASSOCIATED VIRUS (AAV) AND
RECOMBINANT AAV VECTORS, Douglas M. McCarty,
Samuel M. Young Jr., and Richard J. Samulski
819
INDEXES
Subject Index
ERRATA
An online log of corrections to Annual Review of Genetics chapters
may be found at http://genet.annualreviews.org/errata.shtml
847