Mary K. Campbell
Shawn O. Farrell
international.cengage.com/
Chapter Eleven
Transcription of the Genetic Code: The
Biosynthesis of RNA
Paul D. Adams • University of Arkansas
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Transcription
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Transcription in Prokaryotes
• E. coli RNA Polymerase:
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molecular weight about 500,000
four different types of subunits: α, β , β’, and σ
the core enzyme is α2ββ’
the holoenzyme is α2ββ’σ
the role of the σ subunit is recognition of the promoter locus;
locus
the σ subunit is released after transcription begins
• of the two DNA strands, the one that serves as the template for
RNA synthesis is called the template strand or antisense
strand; the other is called the coding (or nontemplate) strand
strand
or sense strand
• the holoenzyme binds to and transcribes only the template
strand
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The Basics of Transcription
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Coding strand
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Promoter Sequence
• Simplest of organisms contain a lot of DNA that is
not transcribed
• RNA polymerase needs to know which strand is
template strand, which part to transcribe, and where
first nucleotide of gene to be transcribed is
• Promoters-DNA sequence that provide direction for
RNA polymerase
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Promoter Sequence
Promoters typically consist of 40 bp region on the 5'-side of the transcription start site
Two consensus sequence elements:
• The "-35 region", with consensus TTGACA
• The Pribnow box near -10, with consensus TATAAT –
this region is ideal for unwinding - why?
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Chain Initiation
• First phase of transcription is initiation
• Initiation begins when RNA polymerase binds to
promoter and forms closed complex
• After this, DNA unwinds at promoter to form open
complex, which is required for chain initiation
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Initiation and Elongation in Transcription
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Chain Elongation
• After strands separated, transcription bubble of ~17
bp moves down the DNA sequence to be transcribed
• RNA polymerase catalyzes formation of
phosphodiester bonds between the incorp.
ribonucleotides
• Topoisomerases relax supercoils in front of and
behind transcription bubble
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Chain Elongation (Cont’d)
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Chain Termination
• Two types of termination mechanisms:
• intrinsic termination- controlled by specific
sequences, termination sites
• Termination sites characterized by two inverted
repeats
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Chain Termination (Cont’d)
• Other type of termination involves rho (ρ) protein
• Rho-dependent termination sequences cause hairpin
loop to form
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Transcription Regulation in Prokaryotes
• In prokaryotes, transcription regulated by:
• alternative σ factors
• enhancers
• operons
• transcription attenuation
• Alternative σ factors
• Viruses and bacteria exert control over which genes
are expressed by producing different σ-subunits that
direct the RNA polymerase to different genes.
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Control by Different σ Subunits
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Operon
• Operon:
Operon: a group of operator, promoter, and
structural genes that codes for proteins
• the control sites, promoter, and operator genes are
physically adjacent to the structural gene in the DNA
• the regulatory gene can be quite far from the operon
• operons are usually not transcribed all the time
β-Galactosidase,
Galactosidase, an inducible protein
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coded for by a structural gene, lacZ
structural gene lacY codes for lactose permease
structural gene lacA codes for transacetylase
expression of these three structural genes is
controlled by the regulatory gene lacI that codes for a
repressor
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How Does Repression Work
• Repressor protein
made by lacI gene
forms tetramer when it
is translated
• Repressor protein then
binds to operator
portion of operon
• Operator and promoter
together are the
control sites
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Binding Sites On the lac operon
• Lac operon is induced when E. coli has lactose as
the carbon source
• Lac protein synthesis repressed by glucose
(catabolite repression)
• E. coli recognizes presence of glucose by promoter
as it has 2 regions: RNA polymerase binding site,
catabolite activator protein (CAP) binding site
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Binding Sites On lac operon (Cont’d)
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Catabolite Repression
• CAP forms
complex with
cAMP
• Complex binds at
CAP site
• RNA polymerase
binds at available
binding site, and
transcription occurs
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Enhancers
• Certain genes include sequences upstream of
extended promoter region
• These genes for ribosomal production have 3
upstream sites, Fis sites
• Class of DNA sequences that do this are called
enhancers
• Bound by proteins called transcription factors
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Elements of a Bacterial Promoter
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Basic Control Mechanisms in Gene Control
• Control may be inducible or repressive, and these
may be negatively or positively controlled
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Control of the trp operon
• Trp operon codes for a leader sequence (trpL) and five
polypeptides
• The five proteins make up 4 different enzymes that catalyze
the multistep process that converts chorisimate to tryptophan
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Alternative 2˚ structures Can Form in trp
Operon
• These structures can
form in the leader
sequence
• Pause structurebinding between
regions 1 and 2
• Terminator loopbinding between
regions 3 and 4
• Antiterminator
structure- Alternative
binding between
regions 2 and 3
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Attenuation in the trp operon
• Pause structure
forms when
ribosome passes
over Trp codons
when Trp levels are
high
• Ribosome stalls at
the Trp codon when
trp levels are low
and antiterminator
loop forms
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Transcription in Eukaryotes
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Three RNA polymerases are known; each
transcribes a different set of genes and recognizes
a different set of promoters:
• RNA Polymerase I- found in the nucleolus and
synthesizes precursors of most rRNAs
• RNA Polymerase II- found in the nucleoplasm
and synthesizes mRNA precursors
• RNA Polymerase III- found in the nucleoplasm
and synthesizes tRNAs, other RNA molecules
involved in mRNA processing and protein transport
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RNA Polymerase II
• Most studied on the polymerases
• Consists of 12 subunits
• RPB- RNA Polymerase B
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How does Pol II Recognize the Correct
DNA?
• Four elements of the Pol II promoter allow for this
phenomenon
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Initiation of Transcription
• Any protein regulator of transcription that is not itself
a subunit of Pol II is a transcription factor
• Initiation begins by forming the preinitiation
complex
• Transcription control is based here
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General Transcription Initiation Factors
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Transcription Order of Events
• Less is known about
eukaryotes than
prokaryotes
• The phosphorylated
Pol II synthesizes
RNA and leaves the
promoter region
behind
• GTFs are left at the
promoter or
dissociate from Pol II
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Elongation and Termination
• Elongation is controlled by:
• pause sites, where RNA Pol will hesitate
• anti-termination, which proceeds past the normal
termination point
• positive transcription elongation factor (P-TEF) and
negative transcription elongation factor (N-TEF)
• Termination
• begins by stopping RNA Pol; the eukaryotic
consensus sequence for termination is AAUAAA
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Gene Regulation
• Enhancers and silencers- regulatory sequences
that augment or diminish transcription, respectively
• DNA looping brings enhancers into contact with
transcription factors and polymerase
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Eukaryotic Gene Regulation
• Response elements are enhancers that respond to
certain metabolic factors
• heat shock element (HSE)
• glucocorticoid response element (GRE)
• metal response element (MRE)
• cyclic-AMP response element (CRE)
• Response elements all bind proteins (transcription
factors) that are produced under certain cell
conditions
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Activation of transcription Via CREB and CBP
• Unphosphorylated
CREB does not bind
to CREB binding
protein, and no
transcription occurs
• Phosphorylation of
CREB causes binding
of CREB to CBP
• Complex with basal
complex (RNA
polymerase and
– cAMP-response element
GTFs) activates CRE
CREB – cAMP-response element binding protein
transcription
CBP – CREB-binding protein
PKA – cAMP dependent protein kinase (protein kinase A)
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Response Elements
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Non-Coding RNAs
• As much as 98% of transcriptional output from
human genomes may be comprised of non-coding
RNAs (ncRNA)
• Linked to: regular transcription, gene silencing,
replication, processing of RNA, RNA modification,
translation, protein stabilization, protein translocation
• Two main types: Micro RNA (miRNA), and Small
Interfering RNA (siRNA)
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SiRNAs are formed in away similar miRNA
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Structural Motifs in DNA-Binding Proteins
• Most proteins that activate or
inhibit RNA Pol II have two
functional domains:
• DNA-binding domain
• transcription-activation domain
• DNA-Binding domains have
domains that are either:
• Helix-Turn-Helix (HTH)
• Zinc fingers
• Basic-region leucine zipper
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Helix-Turn-Helix Motif
Hydrogen bonding between amino acids and DNA
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Zinc Finger Motif
• Motif contains 2
cysteines and 2 His
12 amino acids later
• Zinc binds to the
repeats
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Basic Region Leucine Zipper Motif
• Many transcription factors contain this motif, such as
CREB (Biochemical Connections, page 309)
• Half of the protein composed of basic region of
conserved Lys, Arg, and His
• Half contains series of Leu
• Leu line up on one side, forming hydrophobic pocket
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Helical Wheel Structure of Leucine Zipper
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Transcription Activation Domains
• acidic domains- rich in Asp and Glu. Gal4 has
domain of 49 amino acids, 11 are acidic
• glutamine-rich domains- Seen in several
transcription factors. Sp1 has 2 glutamine-rich
domains, one with 39 Glu in 143 amino acids
• proline-rich domains- Seen in CTF-1 (an activator). It
has 84 amino acid domain, of which 19 are Pro
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Post Transcriptional RNA Modification
• tRNA, rRNA, and mRNA are all modified after transcription
to give the functional form
• the initial size of the RNA transcript is greater than the final
size because of the leader sequences at the 5’ end and
the trailer sequences at the 3’ end
• the types of processing in prokaryotes can differ greatly
from that in eukaryotes, especially for mRNA
• Modifications
• trimming of leader and trailer sequences
• addition of terminal sequences (after transcription)
• modification of the structure of specific bases (particularly
in tRNA)
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Posttranscriptional Modification of tRNA
Precursor
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Modification of tRNA
• Transfer RNA- the
precursor of several
tRNAs is can be
transcribed as one long
polynucleotide sequence
• trimming, addition of
terminal sequences,
and base modification
all take place
• methylation and
substitution of sulfur for
oxygen are the two most
usual types of base
modification
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Modification of rRNA
• Ribosomal RNA
• processing of rRNA is primarily a matter of
methylation and trimming to the proper size
• in prokaryotes, 3 rRNAs in one intact ribosome
• in Eukaryotes, ribosomes have 80s, 60s, and 40s
subunits
• base modification in both prokaryotes and eukaryotes
is primarily by methylation
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Modification of mRNA
• Includes the capping
of the 5’ end with an
N-methylated guanine
that is bonded to the
next residue by a 5’ ->
5’ triphosphate.
• Also, 2’-O-methylation
of terminal ribose(s)
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mRNA Modification
• A polyadenylate “tail” that is usually100-200
nucleotides long, is added to the 3’ end before the
mRNA leaves the nucleus
• This tail protects the mRNA from nucleases and
phosphatases
• Eukaryote genes frequently contain intervening base
sequences that do not appear in the final mRNA of
that gene product
• Expressed DNA sequences are called exons
• Intervening DNA sequences that are not expressed
are called introns
• These genes are often referred to as split genes
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Organization of Split Genes in Eukaryotes
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The Splicing Reaction
• Exons are
separated by
intervening intron
• When the exons
are spliced
together,a lariat
forms in the intron
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Ribozymes
• The first ribozymes discovered included those that
catalyze their own self-splicing
• More recently, ribozymes have been discovered that
are involved in protein synthesis
• Group I ribozymes
• require an external guanosine
• example: pre-rRNA of the protozoan Tetrahymena
(next screen)
• Group II ribozymes
• display a lariat mechanism similar to mRNA splicing
• no requirement for an external nucleotide
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Self-splicing of pre-rRNA
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