JB Accepts, published online ahead of print on 3 November 2006
J. Bacteriol. doi:10.1128/JB.01356-06
Copyright © 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Naturally occurring adenines within mRNA coding sequences affect ribosome
binding and expression in Escherichia coli
Authors: Jay E. Brock1, Robert L. Paz2, Patrick Cottle, and Gary R. Janssen*
Running Title: Adenine stimulation of gene expression
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Key Words: translation initiation, ribosome binding, gene expression, adenine
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stimulation, translation enhancer
Author Affiliation: 1Department of Microbiology
Miami University
32 Pearson Hall
Oxford, Ohio 45056 USA
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Phone: 513-529-5448
Fax: 513-529-2431
Email: [email protected]
Address for correspondence:
Gary Janssen
Department of Microbiology
Miami University
32 Pearson Hall
Oxford, Ohio 45056 USA
Phone: 513-529-5422
Fax: 513-529-2431
Email: [email protected]
2
The Fuqua School of Business
Duke University
Box 90120
Durham, NC 27708-0120
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*corresponding author
Abstract
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Translation initiation requires the precise positioning of a ribosome at the start
codon. The major signals of bacterial mRNA that direct the ribosome to a translational
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start site are the Shine-Dalgarno (SD) sequence, within the untranslated leader, and the
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start codon. Evidence for many non-SD-led genes in prokaryotes provides motive for
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studying additional interactions between ribosomes and mRNA that contribute to
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translation initiation. A high incidence of adenines have been reported downstream of
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the start codon for many E. coli genes and addition of downstream adenine-rich
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sequences increases expression from several genes in E. coli. We describe here site-
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directed mutagenesis of the E. coli aroL, pncB and cysJ coding sequences to assess the
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contribution of naturally occurring adenines to in vivo expression and in vitro ribosome
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binding from mRNAs with different SD-containing untranslated leaders. Base
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substitutions that decreased the downstream adenines by one or two nucleotides
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decreased expression significantly from aroL-, pncB- and cysJ-lacZ fusions; mutations
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increasing downstream adenines by one or two nucleotides increased expression
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significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition
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(toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo
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expression correlated closely with changes in in vitro ribosome binding strength. Our
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data are consistent with a model in which downstream adenines influence expression
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through their effects on the mRNA-ribosome association rate and the amount of ternary
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complex formed. This work provides evidence that adenine-rich sequence motifs might
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serve as a general enhancer of E. coli translation.
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Introduction
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Initiation of bacterial protein synthesis requires the selection of an mRNA’s
translation initiation region (TIR) and initiator tRNA (fMet-tRNAfMet) by the 30S
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ribosomal subunit aided by the three initiation factors (IF1, IF2 and IF3). Initiation is the
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rate-limiting step of translation, and the formation of ribosome/initiator tRNA/mRNA
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ternary complexes is influenced by sequence and structural motifs in and around the
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mRNA’s ribosome binding site (RBS) (33). Features of the mRNA RBS contributing to
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the efficiency of ribosome binding and translation include the start codon (i.e., AUG most
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frequently in Escherichia coli) and the purine-rich Shine-Dalgarno (SD) sequence,
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located within the untranslated leader region (UTR), which base-pairs with the anti-SD
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(ASD) sequence near the 3’ end of 16S rRNA (29, 11). In addition to the start codon and
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SD:ASD interaction, other sequence and structural motifs within mRNA have been
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suggested to influence ribosome binding and translation including mRNA secondary
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structure within the TIR (6, 7), specific translation-enhancing sequences upstream (10,
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21, 37, 12) and downstream (32, 9, 19, 26) to the start codon, upstream pyrimidine-rich
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tracts (1, 42, 38), AU-rich sequences within the UTR (14, 15), and downstream A (3) and
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CA-rich tracts (16). In addition, an increasing number of non-SD-led genes (2) and genes
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encoding mRNA lacking a 5’-untranslated leader region (leaderless mRNA) (13, 18) are
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being identified, raising the potential for novel sequence and structural motifs within the
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coding sequence that contribute to the formation of translation initiation complexes.
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Statistical analysis of E. coli translational start sites (24, 27) revealed that the
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region downstream of the initiation codon for several genes contain CA- and/or A-rich
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sequences. In a recent analysis of nucleotide sequences around the boundaries of all open
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reading frames in E. coli, nucleotide biases were observed immediately downstream of
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the initiation codon and the two most frequent second codons, AAA and AAU, were
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found to enhance translational efficiency (26). In addition to these studies, adenine-rich
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sequences have been proposed to enhance translation in E. coli, as demonstrated by the
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increased expression observed for the human gamma interferon (γ-IFN) and
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chloramphenicol acetyltransferase (cat) genes after the addition of A-rich motifs
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downstream of the initiation codon (3).
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The insertion of CA multimers immediately downstream of the lacZ initiation
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codon results in an increase in gene expression (16, 30; G. R. Janssen, unpublished data).
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CA nucleotides, inserted downstream of the start codon for leaderless or SD-leadered
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mRNAs, exerted a greater stimulation when close to the initiation codon and exhibited a
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dose-dependent increase in expression with an increasing number of CA repeats.
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Increased expression was observed also after the addition of CA repeats to SD-leadered
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and leaderless neo, kan and gusA genes indicating that CA-rich sequences might serve as
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a general enhancer of expression for leadered and leaderless mRNAs in E. coli (16).
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The work of Chen et al. (3) and Martin-Farmer and Janssen (16) demonstrate that
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the addition of adenines and CA repeats, respectively, can dramatically increase gene
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expression. The experiments described here investigate the influence of naturally
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occurring downstream adenines on in vivo expression and in vitro ribosome binding. The
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mRNAs encoded by the E. coli aroL, pncB and cysJ genes include SD-containing
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untranslated leaders and adenines downstream of their AUG start codons; site-directed
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mutagenesis was used to decrease or increase the A-richness in order to generate putative
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“down” or “up” mutations, respectively. The putative “down” mutations in aroL, pncB
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and cysJ mRNAs decreased both in vivo expression and in vitro ribosome binding,
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whereas putative “up” mutations in the aroL and cysJ mRNAs increased both in vivo
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expression and in vitro ribosome binding. Using toeprint, filter binding and expression
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assays, our data show that downstream adenines contribute significantly to the rate and
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amount of ternary complex formed as well as the in vivo expression levels for the aroL,
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pncB and cysJ genes even in the presence of a canonical SD sequence.
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Materials and Methods
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Bacterial strains. E. coli DH5α [F-, phi80d, lacZM15∆ (lacZYA-argF), U169, recA1,
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endA1, hsdR17 (rk-, mk+), supE44, lambda-, thi-1, gyrA, relA1] was used as the host
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strain for all plasmid constructs. E. coli RFS859 [F-, thr-1, araC859, leuB6, ∆lac74, tsx-
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274, lambda-, gyrA111, recA11, relA1, thi-1], a lac-deletion strain (28), was used as the
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host for the expression and assay of β-galactosidase activity from lacZ reporter genes. E.
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coli K12 total genomic DNA was used for the isolation of wild type (WT) aroL, cysJ and
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pncB gene fragments. E. coli MRE600 (39) was used for the isolation of 30S ribosomal
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subunits.
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Reagents and recombinant DNA procedures. Radiolabelled nucleotides, [γ-32P]ATP
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(6,000 Ci/mmol; 150 mCi/ml) and [α-32P]CTP (3,000 Ci/mmol; 10 mCi/ml), were
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purchased from Perkin Elmer. Oligonucleotides were synthesized using a Beckman
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1000M DNA synthesizer or purchased from commercial suppliers. Restriction
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endonucleases, T4 DNA ligase, T4 polynucleotide kinase, T4 DNA polymerase and T7
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RNA polymerase were obtained from New England Biolabs. Pfu DNA polymerase was
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obtained from Stratagene. AMV reverse transcriptase was obtained from Life Sciences
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and RNase-free DNase I was purchased from Ambion. All enzymes were used according
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to the manufacturer’s recommendations. Plasmid isolation, E. coli transformations and
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other DNA manipulations were carried out in a standard manner (25).
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Mutant Constructions. The aroL-, cysJ- and pncB-lacZ fusions were constructed
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containing either the lacZ untranslated leader or the gene’s natural untranslated leader
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and the first sixteen codons of the coding sequence fused to a lacZ reporter gene.
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Transcription of the lac fusions was provided by the E. coli lac promoter. Plasmids
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(pBR322-derived) containing the aroL-, cysJ- and pncB-lacZ fusions were used as
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templates for site-directed mutagenesis in which one oligonucleotide primer contained
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the desired mutation(s) (Table 1) and the other primer annealing within the lacZ coding
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sequence. After amplification, the PCR product was trimmed with appropriate restriction
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enzymes to facilitate cloning, ultimately producing identical plasmids varying only by the
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single or double nucleotide mutations made within the coding sequence as shown in
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Table 1. All DNA regions generated by PCR amplification were verified by DNA
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sequencing.
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β-galactosidase assays. Triplicate cultures of plasmid-containing strains were grown in
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2XYT (per liter, 16g of Difco Bacto Tryptone, 10 g of Difco Bacto yeast extract, 10 g of
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NaCl, pH 7.4) supplemented with 200 µg/ ml ampicillin and 0.2 mM IPTG, at 37°C to an
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O.D.600 of 0.4-0.6 and quick chilled on ice. Triplicate β-galactosidase assays (17) were
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performed with each of the triplicate cultures.
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Primer extension inhibition (toeprinting). The mRNAs used in primer extension
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inhibition (toeprint) experiments were generated in vitro with T7 RNA polymerase.
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DNA templates for T7 transcription were prepared by PCR amplification of miniprep
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plasmid DNA. Oligonucleotide primers T7lac.lead (5’-
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ggaattctaatacgactcactatagaattgtgagcggataacaatttc-3’) and lac.comp2 (5’-
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attaagttgggtaacgccag-3’) were used to generate DNA fragments containing the T7
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promoter, lac leader and cysJ sequences (with or without mutations) fused to lacZ from
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the respective template plasmids. T7pncB.lead (5’-
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ctaatacgactcactatagttcctgaagatgtttattgtac-3’) and lac.comp2 were used to generate DNA
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fragments containing the T7 promoter, pncB leader and pncB sequences (with or without
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mutations) fused to lacZ. T7aroL.lead (5’-
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ggaattctaatacgactcactatagattgagattttcactttaagtgg-3’) and lac.comp2 were used to generate
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DNA fragments containing the T7 promoter, aroL leader and aroL sequences (with or
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with out mutations) fused to lacZ. After gel purification of the resulting PCR-generated
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fragments, transcription reactions [15 mM DTT, 4 mM NTPs, 40 mM Tris-HCL (pH7.9),
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20 mM MgCl2, 1 µg template DNA, and 1 µl T7 RNA polymerase (NEB; 500 U/ µl); in a
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volume of 20 µl] were carried out at 37°C for 1 hour. This was followed by 1 µl of
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RNase-free DNase I [2 U/µl] for 30 minutes at 37°C. In vitro synthesized mRNA was
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then gel purified on 6% polyacrylamide 7M urea denaturing gels. After UV shadowing
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and elution of the mRNA with 0.2% SDS, 0.005M EDTA and 0.5M NH4OAc, mRNA
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was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with
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0.2 M NH4OAc and 2.5 volumes of EtOH.
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Isolation of E. coli 30S ribosomal subunits and toeprint assays were done as
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previously described (16). Briefly, 9 µl reactions containing mRNA (44nM) with a 32P-
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labelled primer annealed to the 3’ end, 30S ribosomal subunits, tRNAfMet (1:10:20 ratio)
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and 1XSB [60mM NH4Cl, 10mM Tris-OAc (pH 7.4), 10mM MgOAc, 6mM beta-
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mercaptoethanol] were incubated at 37°C for 30 minutes or the indicated times.
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Reactions were placed on ice and 1µl of reverse transcriptase (1 U/µl) was added
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followed by a 37°C incubation for 10 minutes. Reactions were precipitated with 40 µl of
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0.3M NaOAc and 2.5 volumes of EtOH for at least 2.5 hours at -20°C and
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electrophoresed on 6% polyacrylamide (7M urea) gels. Dideoxy sequencing reactions
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mapped toeprint signals to the +16 position of mRNA relative to the first base of the start
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codon (+1). Toeprint signals were quantified with a Molecular Dynamics
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PhosphorImager (Storm 800) and expressed as relative toeprint complexes (RTC)
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[toeprint pixel value/(toeprint pixel value + full-length pixel value)] or [toeprint pixel
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value/(toeprint pixel value + full-length pixel value + upstream signal pixel value)] for
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aroL RTCs. Comparisons of RTCs between different mRNAs were made from reactions
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electrophoresed on the same gel.
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Filter binding assays. mRNA used in filter binding assays was synthesized in a 5 µl
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reaction containing 5mM DTT, 2mM NTPs, 14mM MgCl2, 0.5 µl 10X T7 RNA
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polymerase buffer (NEB), 0.05 µg template DNA, 0.35 µl T7 RNA polymerase (NEB;
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500 U/ µl) and 2.3 µl [α-32P]CTP. Filter binding reactions (10 µl) containing
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radiolabeled mRNA (44nM), 30S ribosomal subunits, tRNAfMet (1:10:20 ratio) and 1XSB
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were incubated for various amounts of time at 37°C. Reaction mixtures were then diluted
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to 500 µl with chilled 1XSB and filtered through a nitrocellulose membrane (0.45-µm
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pore size) in a Mini-Fold slot blot manifold (Schleicher and Schuell) followed by the
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washing of each well with 3 ml of 1XSB. Membranes were then dried at room
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temperature and samples cross-linked to the membranes by UV light (Fisher Scientific,
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FB-UVXL-1000). The amount of complex bound to the membrane was quantified with a
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Molecular Dynamics PhosphorImager (Storm 800); standard curves converted pixel
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values to picomoles, as previously described (4), and data expressed as picomoles of
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mRNA bound.
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Results
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All base substitutions in the aroL, pncB and cysJ genes were made via sitedirected mutagenesis and maintain the natural amino acid sequence; putative “down”
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mutations decrease the downstream adenine content whereas putative “up” mutations
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increase the downstream adenine content (Table 1). The effects of the mutations on gene
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expression were assessed by translational fusions of aroL, pncB and cysJ gene fragments
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to an E. coli lacZ reporter gene followed by β-galactosidase assays. Toeprint and filter
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binding assays were used to compare the rate of ternary complex formation, defined here
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as the amount of complex formed (ribosomes/tRNA/mRNA) over time, for aroL, pncB
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and cysJ mRNAs with or without mutations that alter the downstream adenine content
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(Table 1). In toeprint assays, variations in a specific RTC might be observed between
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different assays but the correlation between wild type and mutant binding strengths were
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consistent and reproducible. These assays were performed in an effort to correlate the in
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vitro ribosome binding strength of aroL, pncB and cysJ mRNAs with the in vivo
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expression data.
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Downstream adenines of aroL
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Transcription of the aroL gene, encoding shikimate kinase II, results in an mRNA
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with a 124 nucleotide leader containing an optimally spaced (7-9 nucleotides), canonical
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SD sequence (5). Mutations in aroL’s coding sequence altering the downstream adenine
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content are shown in Table 1.
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i. The effect of aroL downstream adenines on in vivo expression
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Comparisons of β-galactosidase activity between cells containing the wild type
(pAaroL.WT) or the putative “down” aroL-lacZ constructs revealed that the A→G
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substitutions in codons two and three (pAaroL.DN1) decreased lacZ expression 72%;
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incorporation of the individual substitutions resulted in a 15% (pAaroL.DN1a) and 44%
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(pAaroL.DN1b) decrease in lacZ expression (Figure 1-A). The putative “up” aroL-lacZ
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construct revealed that the U→A substitution at codon four (pAaroL.UP1) increased lacZ
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expression 38% (Figure 1-A). These results indicate that the downstream adenines
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influence aroL expression significantly, despite the presence of a canonical SD:ASD
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interaction.
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ii. The effect of aroL downstream adenines on in vitro ribosome binding
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Toeprint assays with aroL-lacZ mRNA and 30S subunits revealed a tRNA-
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dependent signal corresponding to position +16 relative to the A (+1) of the AUG start
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codon (Figure 2-A, lanes 7-9). Phosphorimage analysis of toeprint signal intensity
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revealed a 97% reduction from the putative “down” mutant mRNA (Figure 2-B, lane 15)
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compared to wild type (Figure 2-B, lane 14). Incorporation of the U→A putative “up”
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mutation at codon four increased the toeprint signal 278% (Figure 2-B, lane 16)
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compared to wild type (Figure 2-B, lane 14). The toeprint results, when taken with the
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aroL-lacZ expression levels, show a positive correlation between the downstream adenine
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content, in vitro ribosome binding and in vivo expression levels.
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iii. The effect of aroL downstream adenines on the rate and amount of ternary complex
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formed
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Incorporation of the A→G putative “down” mutations at aroL’s second and third
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codons (Figure 3-A, lanes 10-19) resulted in a reduced rate and amount of ternary
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complex formation compared to wild type (Figure 3-A, lanes 1-9), as quantified in Figure
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3-B. Incorporation of the U→A putative “up” mutation at aroL’s fourth codon (Figure 3-
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A, lanes 20-29) resulted in an increased rate and amount of ternary complex formation
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compared to wild type (Figure 3-A, lanes 1-9), as quantified in Figure 3-B. The
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significant amount of toeprint signal at t=0 (Figure 3-A and B) reflects the complex
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formed and cDNA produced during the ten minute incubation time for reverse
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transcriptase. An additional uncharacterized ternary complex signal is observed within
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aroL’s untranslated leader and is noted with an asterisk (*) in Figure 3-A. Because this
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signal occurs upstream to the aroL toeprint signal (arrow, Figure 3-A), it was included
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with the full length signal for phosphorimage quantification of RTC values in Figure 3-B.
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Calculation of RTC values without the additional band (*) resulted in higher RTC values
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but the line slopes, relative to each other, were basically unchanged (data not shown).
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In order to get a more accurate assessment of complex formation at earlier time
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points, filter binding assays were used. Filter binding assays measure complex formation
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based on the amount of mRNA bound by ribosomes when filtered through a
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nitrocellulose membrane. Incorporation of the A→G putative “down” mutations at
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aroL’s second and third codons reduced the rate and amount of ternary complex formed,
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whereas the U→A putative “up” mutation at codon four increased the amount of ternary
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complex formed compared to wild type (Figure 3-C). Ternary complex formation with
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the “up” mutant demonstrated a similar rate to the wild type during the first 60 seconds of
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incubation, after which the amount of product formed continued to increase with the “up”
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mutant but not with the wild type (Figure 3-C). The toeprint and filter binding results
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provide evidence that downstream adenines enhance the rate and/or amount of ternary
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complex formation with aroL mRNA.
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Downstream adenines of pncB
Transcription of the pncB gene, encoding a nicotinic acid
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phosphoribosyltransferase, results in an mRNA with a 58 nucleotide untranslated leader
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containing a canonical SD sequence with suboptimal spacing (12 nucleotides) from the
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start codon (40) and identical second and third codons to aroL. Mutations in pncB’s
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coding sequence altering the downstream adenine content are shown in Table 1.
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i. The effect of pncB downstream adenines on in vivo expression
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Comparisons of β-galactosidase activities between cells containing the wild type
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(pApncB.WT) or the putative “down” pncB-lacZ constructs revealed that the A→G
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substitutions in codons two and three (pApncB.DN1) decreased lacZ expression 99.9%;
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incorporation of the individual substitutions resulted in an 89% (pApncB.DN1a) and 92%
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(pApncB.DN1b) decrease in lacZ expression (Figure 1-B). The putative “up” pncB-lacZ
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construct revealed that the U→A substitutions in codons five and six (pApncB.UP1)
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unexpectedly decreased lacZ expression 67% (Figure 1-B).
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To determine if altering the pncB downstream adenine content has a similar effect
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on expression irrespective of the untranslated leader sequence, analogous pncB-lacZ
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coding sequence fusions were placed downstream of the E. coli lacZ untranslated leader
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containing a canonical SD sequence with optimal spacing (7 nucleotides) from the start
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codon. Putative “down” mutations in codons two and three of the lac-leadered pncB-lacZ
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mRNA reduced gene expression 87% relative to wild type (data not shown). These
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results indicate that the properly spaced canonical SD sequence of the lac leader could
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not fully compensate for the loss of downstream adenines.
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ii. The effect of pncB downstream adenines on in vitro ribosome binding
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Toeprint assays with pncB-lacZ mRNA and 30S subunits revealed a tRNAdependent signal corresponding to position +16 relative to the A (+1) of the AUG start
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codon (Figure 2-A, lanes 1-3). pncB-lacZ mRNA containing putative “down” mutations
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at codon three (Figure 2-B, lane 4) or codons two and three (Figure 2-B, lane 5) nearly
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eliminated ribosome binding (toeprint signal reduced >95%) compared to wild type
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(Figure 2-B, lane 3). Incorporation of the putative “up” mutations at codons five and six
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resulted in a 62% reduction in toeprint signal intensity (Figure 2-B, lane 6) compared to
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wild type (Figure 2-B, lane 3). The toeprint results, when taken with the pncB-lacZ
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expression levels, show a positive correlation between in vitro ribosome binding and the
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in vivo expression levels.
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iii. The effect of pncB downstream adenines on the rate and amount of ternary complex
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formed
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Incorporation of the A→G putative “down” mutation at pncB’s third codon
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(Figure 4-A, lanes 9-16) resulted in a dramatically reduced rate and amount of ternary
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complex formation when compared to wild type (Figure 4-A, lanes 1-8), as quantified in
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Figure 4-B. Bands observed in the absence of tRNA and ribosomes are artifacts likely
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due to structures formed within the mRNA. When analyzed by filter binding assays,
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incorporation of the A→G putative “down” mutation at pncB’s third codon resulted in a
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reduced rate and amount of ternary complex formation compared to wild type (Figure 4-
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C). The toeprint and filter binding results provide evidence that downstream adenines
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contribute to the rate and amount of ternary complex formation with pncB mRNA.
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Downstream adenines of cysJ
The E. coli cysJ gene, encoding a NADPH-sulfite reductase flavoprotein
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component (22), has different second and third codons from aroL and pncB. Cloning a
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DNA fragment encoding the cysJ untranslated leader and a fragment of cysJ coding
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sequence into a multicopy plasmid caused a dramatic reduction in plasmid copy number
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(data not shown); therefore, cysJ-lacZ coding sequence fusions were placed downstream
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of the 38 nucleotide lacZ untranslated leader containing a canonical SD sequence with
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optimal spacing (7-9 nucleotides) from the cysJ start codon. Mutations in cysJ’s coding
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sequence altering the downstream adenine content are shown in Table 1.
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i. The effect of cysJ downstream adenines on in vivo expression
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Comparisons of β-galactosidase activities between cells containing the wild type
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(pAZcysJ.WT) or the putative “down” (pAZcysJ.DN1) cysJ-lacZ construct revealed that
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the third codon A→G base substitution resulted in a 77% decrease in lacZ expression
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(Figure 1-C). The putative “up” cysJ-lacZ constructs revealed that the G→A
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substitutions in codons two and four (pAZcysJ.UP1) increased lacZ expression 525%;
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incorporation of the individual substitutions resulted in a 174% (pAZcysJ.UP1a) and
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225% (pAZcysJ.UP1b) increase in expression (Figure 1-C).
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ii. The effect of cysJ downstream adenines on in vitro ribosome binding
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Toeprint assays with cysJ-lacZ mRNA and 30S subunits revealed a tRNA-
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dependent signal corresponding to position +16 relative to the A (+1) of the AUG start
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codon (Figure 2-A, lanes 4-6). Phosphorimage analysis of toeprint signal intensity
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revealed an 86% reduction from the putative “down” mutant (Figure 2-B, lane 10)
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compared to wild type (Figure 2-B, lane 9). Incorporation of putative “up” mutations at
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276
298
codons two and four resulted in a 202% increase in toeprint signal intensity (Figure 2-B,
299
lane 11) compared to wild type (Figure 2-B, lane 9). The toeprint results, when taken
300
with the cysJ-lacZ expression levels, show a strong correlation between the downstream
301
adenine content, in vitro ribosome binding and in vivo expression levels.
302
iii. The effect of cysJ downstream adenines on the rate and amount of ternary complex
303
formed
304
D
E
Incorporation of the A→G putative “down” mutation at cysJ’s third codon (Figure
5-A, lanes 10-18) resulted in a reduced rate and amount of ternary complex formation
306
compared to wild type (Figure 5-A, lanes 1-9), as quantified in Figure 5-B. Incorporation
307
of the G→A putative “up” mutations at cysJ’s second and fourth codons (Figure 5-A,
308
lanes 19-27) resulted in an increased rate and amount of ternary complex formation
309
compared to wild type (Figure 5-A, lanes 1-9), as quantified in Figure 5-B.
310
Phosphorimage analysis of the full length, run-off signal yielded consistently high pixel
311
values, resulting in low RTC values; however, the rate comparisons between mutant and
312
WT mRNAs still correlated well with results visualized by autoradiography. When
313
analyzed by filter binding assays, incorporation of the A→G putative “down” mutation at
314
cysJ’s third codon resulted in a similar rate and amount of ternary complex formation as
315
with wild type mRNA during the first three minutes of incubation; after three minutes,
316
the rate and amount of ternary complex formed was reduced compared to wild type
317
(Figure 5-C). Incorporation of the G→A putative “up” mutations at cysJ’s second and
318
fourth codons resulted in an increased rate and amount of ternary complex formation
319
compared to wild type, with the most significant difference in rate occurring within the
320
initial three minutes of binding (Figure 5-C). The toeprint and filter binding results
E
C
C
A
16
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T
P
305
321
provide evidence that downstream adenines enhance the rate and amount of ternary
322
complex formed with cysJ mRNA.
T
P
E
C
C
A
17
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D
E
Discussion
323
324
The correlation of adenine-rich regions with efficient translation in E. coli was
first noted by Dreyfus (8), with later studies reporting stimulatory effects on translation
326
by the addition of adenines (3, 16, 30). We report here that adenines occurring naturally
327
downstream of the initiation codon can contribute significantly to ribosome binding and
328
expression in E. coli. Our data show that downstream adenines can exert major effects
329
on expression even in the presence of canonical SD sequences. Demonstration that
330
downstream nucleotides can function as major effectors of expression is especially
331
interesting in light of a recent report (2) that non-SD led genes, including leaderless
332
mRNAs, are as common as SD-led genes in prokaryotes. The absence of a SD sequence
333
requires that other sequence or structural features of the mRNA help direct the ribosome
334
to the correct initiation site. From work described here, the position and number of
335
downstream adenines may contribute to the ribosome binding strength of mRNAs with,
336
and possibly without, canonical SD sequences.
337
D
E
T
P
E
C
C
A
Our experiments investigate the influence of naturally occurring downstream
338
adenines on in vitro ribosome binding and in vivo expression for the E. coli aroL, pncB
339
and cysJ mRNAs. When compared to the wild type mRNAs, the putative “down”
340
mutations significantly decreased in vitro ribosome binding and in vivo expression;
341
putative “up” mutations in aroL and cysJ significantly increased in vitro ribosome
342
binding and in vivo expression. These data are consistent with earlier reports whereby A-
343
rich sequences added immediately downstream of the start codon stimulated expression
344
from several natural and heterologous genes in E.coli (16, 3). While the pncB “down”
345
mutations demonstrate clearly that adenines in codons two and three are important for
18
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325
346
expression, it is unclear why the putative “up” mutations in codons five and six did not
347
increase expression; possible explanations are that the substitutions reside too far from
348
the start codon (i.e., compared to codons two and four for cysJ and aroL ”up” mutations)
349
or the mutations resulted in a secondary structure that reduced access of a ribosome to the
350
initiation region.
351
Downstream adenines affect rate and amount of ternary complex formed.
352
D
E
Using toeprint and filter binding assays as two independent approaches to
estimate ribosome binding, we observed a strong correlation between the downstream
354
adenine content, in vivo expression and the rate and amount of in vitro ribosome binding,
355
suggesting strongly that the variations in expression were mediated through the observed
356
effects on ribosome binding. Toeprint assays revealed also that aroL, pncB and cysJ
357
ternary complexes, once formed, did not dissociate in the presence of a competitor
358
mRNA (data not shown), in agreement with a previous report that ternary complexes are
359
stable and irreversible in the presence of competitor mRNA (31). Therefore, downstream
360
adenines appear to influence expression through their affect on mRNA-ribosome
361
association and not by preventing ternary complex dissociation.
362
Possible contributions of downstream adenines to ribosome binding and expression
363
Comparison of the codons resulting from aroL, pncB and cysJ mutagenesis to an
364
E. coli genomic codon usage table (GenBank) suggests that the effects on expression are
365
not due to the introduction of rare codons or to low tRNA availability. Also,
366
conservation of the wild type amino acid sequence ensured that expression differences
367
from the mutant constructs were not the result of an altered peptide sequence.
368
Furthermore, the strong correlation between in vitro ribosome binding and in vivo
E
C
C
A
19
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T
P
353
369
expression, for both wild type and mutants, suggests the downstream adenines exert their
370
affect at initiation rather than elongation.
371
It is possible that the adenine effects seen here are due to structure within the TIR.
It has been reported that secondary structure within the TIR may inhibit expression,
373
whereas a more open TIR with less structure allows increased expression (6, 7). In an
374
effort to explore the possible contribution of structure to our results, various lengths of
375
the cysJ-, pncB- and aroL-lacZ mRNAs, with or without mutations, were subjected to
376
MFOLD analysis (43) for prediction of mRNA secondary structures by energy
377
minimization. While slight variations in the predicted structures resulted from the
378
downstream base substitutions, a general correlation of more closed or open structures
379
with respective decreases or increases in expression and ribosome binding was not
380
observed (data not shown). In addition, comparing the signals from reverse transcriptase
381
pausing or terminating at inhibitory secondary structures during toeprint assays does not
382
suggest significant differences in structure between wild type and mutant mRNAs
383
(Figures 3-5). Another possibility involving structure is the formation of adenine-rich
384
pseudoknots, described previously as high-affinity RNA ligands to 30S subunits and
385
ribosomal protein S1 (23). While a contribution by downstream adenines to a stimulatory
386
secondary structure has not been discounted, we do not have any data to support this
387
model.
388
D
E
T
P
E
C
C
A
It is also possible that downstream adenines, perhaps in association with
389
pyrimidines (16), provide a region within the RBS that is recognized specifically by
390
ribosome-associated proteins that promote initiation complex formation. For example,
391
the 30S subunit protein S1 is a strong RNA binding protein reported to concentrate
20
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372
mRNA near the ribosome decoding site (36, 35, 1). Previous studies revealed the binding
393
of S1 to various nucleotide motifs within the untranslated leader including an AU-rich
394
omega sequence (10, 1, 14, 15), a CAU-rich omega-like sequence (38) and a (CAA)n
395
repeat (38). From these data it is conceivable that S1 could bind downstream A-rich
396
regions for a more efficient delivery of mRNA to the ribosome decoding site; however,
397
there has been no direct evidence that S1 binds specific nucleotide motifs downstream of
398
the start codon. Interaction between downstream A-rich regions and 16S rRNA might
399
also contribute to mRNA-ribosome association. In possible support of this, poly (A)
400
RNA formed crosslinks to 16S rRNA nucleotides 1394-1399, located adjacent to the
401
ribosomal P-site (34).
402
D
E
T
P
E
C
Crystal structures of ribosome:mRNA complexes provide evidence that mRNA
403
positions -3 to +10 (with the A of the AUG start codon as +1) are wrapped closely around
404
the neck of the 30S subunit, with most ribosome contacts involving the mRNA backbone
405
rather than its bases (41). However, these crystal structures represent stable complexes
406
and lend little information on interactions occurring during ribosome loading that lead to
407
a stable complex.
408
C
A
Recent experiments from our lab also suggest contact between a natural adenine-
409
rich sequence of a leaderless mRNA and proteins associated with E. coli 30S subunits. In
410
this work, leaderless mRNA encoded by the cI gene of bacteriophage lambda, with a
411
photoactivatable 4-thio uridine in the AUG start codon, has been crosslinked to 30S
412
subunit proteins (J. E. Brock and G. R. Janssen, unpublished data). Crosslinking is
413
inhibited, however, by a competitor cI mRNA that lacked a start codon but contains the
414
natural adenine-rich sequence immediately downstream of the start codon position. Also,
21
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392
415
the same cI competitor mRNA inhibits ribosome binding to wild type cI mRNA in
416
toeprint assays suggesting that the adenines contribute to an interaction between mRNA
417
and components of the ribosome that facilitate ternary complex formation. Additional
418
support for downstream adenine stimulation of leaderless mRNA translation in E. coli is
419
provided by a report (16) where addition of CA multimers increased expression from
420
leaderless lacZ, gusA and neo mRNAs.
421
Implications and application of downstream adenines for expression
T
P
Although the mechanistic details for adenine-enhanced ribosome binding and
423
expression remain to be determined, their ability to stimulate translation from leaderless
424
mRNA (16) indicates that an untranslated leader or SD:ASD interaction is not required.
425
Even in the presence of a canonical SD:ASD interaction, downstream adenines
426
significantly influenced ribosome binding and expression from aroL, pncB and cysJ
427
mRNAs. The first five codons of coding sequence are protected by a ribosome during
428
formation of an initiation complex and thereby represent potential contacts that could
429
contribute to an mRNA’s ribosome binding strength; however, constraints imposed by
430
the encoded amino acid sequence has made it difficult to consider sequence-specific
431
translation enhancers in this region of the mRNA. Genetic code degeneracy and “wobble
432
position” variability, however, could allow for a bias towards adenines within the first
433
few codons while imposing minimal constraints on the encoded amino acids.
434
Downstream adenines, possibly in conjunction with the start codon or other features of
435
the mRNA, might present a recognition motif (sequence and/or structural) to a
436
component of the translational machinery and contribute importantly to the ribosome
E
C
C
A
22
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422
D
E
437
binding strength, with the number and position of adenines allowing for “fine-tuning” of
438
expression.
439
If downstream adenines merely provide an open structure for access of the start
codon to ribosomes, then one might expect adenines to stimulate expression in a variety
441
of translation systems; however, addition of downstream adenines to a neo reporter gene
442
did not increase expression from the G+C-rich, gram-positive Streptomyces lividans (J.
443
M. Day and G. R. Janssen, unpublished data). The possibility that adenine stimulation
444
might not occur in G+C-rich organisms argues that the enhanced ribosome binding and
445
expression is at least partially based on sequence and suggests that adenine stimulation of
446
translation might be limited to E. coli and related organisms. Using crosslinking
447
techniques to identify downstream adenine/ribosome interactions, along with further
448
structural studies, may provide evidence to distinguish between sequence and structural
449
components of adenine stimulation.
450
D
E
T
P
E
C
C
A
Characterization of the adenine effect might allow also for simple engineering of
451
expression levels, up or down, by increasing or decreasing downstream adenines.
452
Identification of mRNA features contributing to ribosome binding and translation, from
453
coding sequences with or without SD sequences (2), is essential to understanding these
454
important stages of gene expression in E. coli and other organisms.
23
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440
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455
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618
619
620
621
622
Table 1. DNA sequence of gene fragments, with or without mutations,
from the aroL, pncB and cysJ genes.
Plasmid a
Sequence b
aroL-lacZ fusions
pAaroL.WT: 5’…tggggaaaacccacgATG
pAaroL.DN1:
…ATG
pAaroL.DN1a:
…ATG
pAaroL.DN1b:
…ATG
pAaroL.UP1:
…ATG
Amino Acid:
M
ACA
ACG
ACG
ACA
ACA
T
pncB-lacZ fusions
pApncB.WT: 5’…caggatactgcgcacctATG
pApncB.DN1:
…ATG
pApncB.DN1a:
…ATG
pApncB.DN1b:
…ATG
pApncB.UP1:
…ATG
Amino Acid:
M
cysJ-lacZ fusions
pAZcysJ.WT: 5’…caggaaacagccATG
pAZcysJ.DN1:
…ATG
pAZcysJ.UP1:
…ATG
pAZcysJ.UP1a:
…ATG
pAZcysJ.UP1b:
…ATG
Amino Acid:
M
a
CAA
CAG
CAA
CAG
CAA
Q
ACA
ACG
ACG
ACA
ACA
T
ACG
ACG
ACA
ACA
ACG
T
CCT
CCT
CCT
CCT
CCA
P
CAA
CAG
CAA
CAG
CAA
Q
ACA
ACG
ACA
ACA
ACA
T
TTC
TTC
TTC
TTC
TTC
F
CAG
CAG
CAA
CAG
CAA
Q
CTT
CTT
CTT
CTT
CTT
P
TTT
TTT
TTT
TTT
TTT
F
GCT
GCT
GCT
GCT
GCA
A
GTC
GTC
GTC
GTC
GTC
V
CTG…3’
CTG…
CTG…
CTG…
CTG…
P
TCT CCT…3’
TCT CCT…
TCT CCT…
TCT CCT…
TCA CCT…
S
P
D
E
T
P
CCA…3’
CCA…
CCA…
CCA…
CCA…
P
E
C
The pA-series of plasmid constructs contain the genes’ natural untranslated
C
A
leader while the pAZ-series contain the lac untranslated leader.
b
Lower case letters represent a portion of the untranslated leader and is shown only
for the wild type (WT) constructs, but is present also in each mutant below it.
The lower case, underlined sequences indicate the presumed SD sequence.
The upper case ATG identifies the translational start site for the aroL, pncB
623
and cysJ coding sequences. The single letter code corresponding to the
624
encoded amino acids is given below the sequence. The upper case, underlined,
625
bold-faced nucleotides indicate base substitutions within the coding region to
626
create putative “down” or “up” mutations.
30
Downloaded from jb.asm.org at Penn State Univ on February 8, 2008
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
Figure Legends
627
Figure 1. The effect of aroL, pncB and cysJ downstream adenines on expression in
629
vivo. β-galactosidase activities from aroL-, pncB- and cysJ-lacZ fusions are compared
630
between cells containing plasmids with or without mutations altering downstream
631
adenine content (see Table 1). A) Plasmid-free cells and cells containing plasmids with
632
the following aroL-lacZ fusions: pAaroL.WT, 100%; pAaroL.DN1, 28%; pAaroL.DN1a,
633
85%; pAaroL.DN1b, 56%; pAaroL.UP1, 138%. B) Plasmid-free cells and cells
634
containing plasmids with pncB-lacZ fusions: pApncB.WT, 100%; pApncB.DN1, 0.1%;
635
pApncB.DN1a, 11%; pApncB.DN1b, 8%; pApncB.UP1, 33%. C) Plasmid-free cells and
636
cells containing plasmids with cysJ-lacZ fusions: pAZcysJ.WT, 100%; pAZcysJ.DN1,
637
23%; pAZcysJ.Up1, 625%; pAZcysJ.UP1a, 274%; pAZcysJ.UP1b, 325%.
638
Figure 2. The effects of aroL, pncB and cysJ downstream adenines on ribosome
639
binding in vitro. Toeprint assays with aroL-, pncB- and cysJ-lacZ mRNAs and 30S
640
subunits compare ribosome binding to mRNAs with or without mutations altering
641
downstream adenine content (see Table 1). The closed arrows indicate the ternary
642
complex-dependent toeprint signals at position +16 relative to the A (+1) of the AUG
643
start codons that result from bound 30S subunits and the asterisk (*) in (B) indicates an
644
uncharacterized ternary complex-dependent signal within the aroL upstream UTR.
645
Bands observed in the absence of tRNA and ribosomes are artifacts likely due to
646
structures formed within the mRNA. A) Lanes: GATC, dideoxy DNA sequence ladder
647
for the pncB template, with the ATG initiation triplet boxed; 1-3, pncB-lacZ WT mRNA;
648
4-6, cysJ-lacZ WT mRNA; 7-9, aroL-lacZ WT mRNA. B) Lanes 1-6 utilize pncB-lacZ
649
mRNAs made from the following templates: (1-3) pApncB.WT, (4) pApncB.DN1b, (5)
D
E
T
P
E
C
C
A
31
Downloaded from jb.asm.org at Penn State Univ on February 8, 2008
628
pApncB.DN1, (6) pApncB.UP1. Lanes 7-11 utilize cysJ-lacZ mRNAs made from the
651
following templates: (7-9) pAZcysJ.WT, (10) pAZcysJ.DN1, (11) pAZcysJ.UP1. Lanes
652
12-16 utilize aroL-lacZ mRNAs made from the following templates: (12-14)
653
pAaroL.WT, (13) pAaroL.DN1, (14) pAaroL.UP1.
654
Figure 3. The effects of aroL downstream adenines on the rate and amount of
655
ternary complex formed. Toeprint and filter binding assays compare ternary complex
656
formation on aroL-lacZ mRNAs with or without mutations altering downstream adenine
657
content. A) In toeprint assays, 30S subunits were incubated with tRNAfMet and aroL-lacZ
658
mRNA for increasing times (minutes). Lanes 1-29 utilize aroL-lacZ mRNAs made from
659
the following templates: (1-9) pAaroL.WT, (10-19) pAaroL.DN1, (20-29) pAaroL.UP1.
660
The closed arrow indicates the position of the ternary complex-dependent toeprint signals
661
at +16 and the asterisk (*) indicates an uncharacterized ternary complex-dependent signal
662
within the upstream UTR. B) The relative toeprint complex (RTC) formed with time was
663
quantified and the data averaged from three independent assays are plotted: ν, aroL-lacZ
664
WT mRNA (pAaroL.WT); Ο, aroL-lacZ “down” mutant mRNA (pAaroL.DN1); ∆, aroL-
665
lacZ “up” mutant mRNA (pAaroL.UP1). C) In filter binding assays, 30S subunits were
666
incubated with tRNAfMet and aroL-lacZ mRNA for increasing time and the amount of
667
bound RNA, averaged from three independent assays, is plotted: ν, aroL-lacZ WT
668
mRNA (pAaroL.WT); Ο, aroL-lacZ “down” mutant mRNA (pAaroL.DN1); ∆, aroL-lacZ
669
“up” mutant mRNA (pAaroL.UP1).
670
Figure 4. The effects of pncB downstream adenines on the rate and amount of
671
ternary complex formed. Toeprint and filter binding assays compare ternary complex
672
formation on pncB-lacZ mRNAs with or without mutations altering downstream adenine
D
E
T
P
E
C
C
A
32
Downloaded from jb.asm.org at Penn State Univ on February 8, 2008
650
content. A) In toeprint assays, 30S subunits were incubated with tRNAfMet and pncB-
674
lacZ mRNA for increasing times (minutes). Lanes 1-16 utilize pncB-lacZ mRNAs made
675
from the following templates: (1-8) pApncB.WT, (9-16) pApncB.DN1b. The arrow
676
indicates the position of the ternary complex-dependent toeprint signals. B) The relative
677
toeprint complex (RTC) formed with time was quantified and the data averaged from
678
three independent assays are plotted: ν, pncB-lacZ WT mRNA (pApncB.WT); Ο, pncB-
679
lacZ “down” mutant mRNA (pApncB.DN1b). C) In filter binding assays, 30S subunits
680
were incubated with tRNAfMet and pncB-lacZ mRNA for increasing time and the amount
681
of bound RNA, averaged from three independent assays, is plotted: ν, pncB-lacZ WT
682
mRNA (pApncB.WT); Ο, pncB-lacZ “down” mutant mRNA (pApncB.DN1b).
683
Figure 5. The effects of cysJ downstream adenines on the rate and amount of
684
ternary complex formed. Toeprint and filter binding assays compare ternary complex
685
formation on cysJ-lacZ mRNAs with or without mutations altering downstream adenine
686
content. A) In toeprint assays, 30S subunits were incubated with tRNAfMet and cysJ-lacZ
687
mRNA for increasing times (minutes). Lanes 1-27 utilize cysJ-lacZ mRNAs made from
688
the following templates: (1-9) pAZcysJ.WT, (10-18) pAZcysJ.DN1, (19-27)
689
pAZcysJ.UP1. The arrow indicates the position of the ternary complex-dependent
690
toeprint signals. B) The relative toeprint complex (RTC) formed with time was
691
quantified and the data averaged from three independent assays are plotted: ν, cysJ-lacZ
692
WT mRNA (pAZcysJ.WT); Ο, cysJ-lacZ “down” mutant mRNA (pAZcysJ.DN1); ∆,
693
cysJ-lacZ “up” mutant mRNA (pAZcysJ.UP1). C) In filter binding assays, 30S subunits
694
were incubated with tRNAfMet and cysJ-lacZ mRNA for increasing time and the amount
695
of bound RNA, averaged from three independent assays, is plotted: ν, cysJ-lacZ WT
D
E
T
P
E
C
C
A
33
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673
696
mRNA (pAZcysJ.WT); Ο, cysJ-lacZ “down” mutant mRNA (pAZcysJ.DN1); ∆, cysJ-
697
lacZ “up” mutant mRNA (pAZcysJ.UP1).
T
P
E
C
C
A
34
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D
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Acknowledgements
698
699
700
701
This work was supported by grant GM065120 from the National Institutes of
Health.
J.E.B. thanks the Miami University Graduate School for a Graduate Student
Achievement Award and the Center for Bioinformatics and Functional Genomics for a
703
summer research fellowship. Special thanks to Eileen Bridge for helpful comments on
704
the manuscript, and Holly Rovito and Michael Day for inspiring discussions and
705
assistance with assay development.
T
P
E
C
C
A
35
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D
E
702
ys
J.U
P1
b
600
pA
Zc
700
ys
J.U
P1
a
C. cysJ
D
N
1
pA
1b
1a
P1
N
N
pn
cB
.U
pn
cB
.D
pn
cB
.D
E
C
pA
pA
pn
cB
.
0
pA
Zc
N
1
pn
cB
.W
T
20
ys
J.U
P1
sJ
.D
pA
pA
40
pA
Zc
Zc
y
ys
J.W
T
RF
S8
59
% Beta-Galactosidase Activity
P1
1b
N
D
1a
N
1
N
ar
oL
.U
pA
ar
oL
.
pA
ar
oL
.D
pA
W
T
ar
oL
.D
pA
85
9
ar
oL
.
pA
RF
S
% Beta-Galactosidase Activity
20
0
B. pncB
120
100
80
60
500
400
300
200
100
0
T
P
D
E
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pA
pA
Zc
RF
S8
59
C
A
% Beta-Galactosidase Activity
Figure 1.
A. aroL
160
140
120
100
80
60
40
Figure 2.
A.
pncB
B.
pncB cysJ aroL
cysJ
aroL
D
E
*
+16
E
C
→
Lane
C T AG
mRNA
tRNAfMet
30S
→
12
++
+ - +
3
+
+
+
4
+
+
-
5
+
+
6
+
+
+
7
+
+
-
8
+
+
C
A
9
+
+
+
Lane
mRNA
tRNAfMet
30S
37
1
+
+
-
2
+
+
3
+
+
+
4
+
+
+
5 6
++
++
++
7
+
+
-
8
+
+
9 10 11 12 13 14 15 16
+ + + + + + + +
+ + + + - + + +
+ + +
- + + + +
\
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T
P
A
T
G
Figure 3.
A.
WT
DN1
UP1
T
P
E
C
→
C
A
Lane
mRNA
tRNAfMet
30S
Time
B.
0.9
0.8
0.7
0.6
0.5
1
+
+
-
2
+
+
3
+
+
+
4
+
+
+
5
+
+
+
6
+
+
+
7
+
+
+
8
+
+
+
9
+
+
+
60 60 0 5 10 15 20 40 60
10
+
+
-
11
+
+
12
+
+
+
60
60
0
13 14
+ +
+ +
+ +
15
+
+
+
16
+
+
+
5 10 15 15
17
+
+
+
18
+
+
+
19
+
+
+
20 21
+ +
+ - +
20 40 60
60
60
22
+
+
+
0
23 24 25 26
+ + + +
+ + + +
+ + + +
27 28 29
+ + +
+ + +
+ + +
5 10 15 15 20 40 60
C.
0.2
0.18
0.16
0.14
0.12
0.1
0.4
0.08
0.3
0.06
0.2
0.04
0.1
0.02
0
0
0
10
20
30
40
50
60
70
0
Time (minutes)
100
200
300
400
Time (seconds
38
500
600
700
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D
E
*
Figure 4.
A.
WT
DN1b
B.
0.25
0.2
0.15
0.1
D
E
0.05
0
0
10
20
30
40
50
60
70
500
600
700
T
P
Time (minutes)
C.
0.16
0.14
E
C
0.12
0.1
0.08
Lane
mRNA
tRNAfMet
30S
1
+
+
2
+
+
-
3
+
+
+
4
+
+
+
Time
60 60 0 5
5
+
+
+
6
+
+
+
7
+
+
+
8
+
+
+
9 10 11 12
+ + + +
- + + +
+ - + +
13 14 15 16
+ + + +
+ + + +
+ + + +
C
A
10 20 40 60
60 60 0
5 10 20 40 60
0.06
0.04
0.02
0
0
39
100
200
300
400
Time (seconds)
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→
Figure 5.
A.
WT
DN1
UP1
Lane
mRNA
tRNAfMet
30S
Time
B.
0.16
0.14
0.12
0.1
0.08
0.06
1
+
+
+
2
+
+
+
3
+
+
+
4
+
+
+
5
+
+
+
6
+
+
+
7
+
+
+
8
+
+
9
+
+
-
0
5 10 15 20 40 60 60 60
10
+
+
+
C
A
E
C
0
T
P
11
+
+
+
5
12
+
+
+
13 14
+ +
+ +
+ +
15 16
+ +
+ +
+ +
17 18
+ +
- +
+ -
10 15 20 40 60 60 60
19
+
+
+
20
+
+
+
0
21 22
+ +
+ +
+ +
23 24 25
+ + +
+ + +
+ + +
26 27
+ +
- +
+ -
5 10 15 20 40 60 60 60
C.
0.12
0.1
0.08
0.06
0.04
0.04
0.02
0.02
0
0
0
10
20
30
40
50
60
70
0
Time (minutes)
100
200
300
400
Time (secon
40
500
600
700
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→