The effects of codon context on in vivo translation speed

pre-printWe developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Low frequency effects of so-called ‘‘silent'' codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 59- and 39- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels

Multiple Promoters Contribute to Swarming and the Coordination of Transcription with Flagellar Assembly in Salmonella▿ †

In Salmonella, there are three classes of promoters in the flagellar transcriptional hierarchy. This organization allows genes needed earlier in the construction of flagella to be transcribed before genes needed later. Four operons (fliAZY, flgMN, fliDST, and flgKL) are expressed from both class 2 and class 3 promoters. To investigate the purpose for expressing genes from multiple flagellar promoters, mutants were constructed for each operon that were defective in either class 2 transcription or class 3 transcription. The mutants were checked for defects in swimming through liquids, swarming over surfaces, and transcriptional regulation. The expression of the hook-associated proteins (FlgK, FlgL, and FliD) from class 3 promoters was found to be important for swarming motility. Both flgMN promoters were involved in coordinating class 3 transcription with the stage of assembly of the hook-basal body. Finally, the fliAZY class 3 promoter lowered class 3 transcription in stationary phase. These results indicate that the multiple flagellar promoters respond to specific environmental conditions and help coordinate transcription with flagellar assembly

Mlc of Thermus thermophilus: a Glucose-Specific Regulator for a Glucose/Mannose ABC Transporter in the Absence of the Phosphotransferase System

We report the presence of Mlc in a thermophilic bacterium. Mlc is known as a global regulator of sugar metabolism in gram-negative enteric bacteria that is controlled by sequestration to a glucose-transporting EII(Glc) of the phosphotransferase system (PTS). Since thermophilic bacteria do not possess PTS, Mlc in Thermus thermophilus must be differently controlled. DNA sequence alignments between Mlc from T. thermophilus (Mlc(Tth)) and Mlc from E. coli (Mlc(Eco)) revealed that Mlc(Tth) conserved five residues of the glucose-binding motif of glucokinases. Here we show that Mlc(Tth) is not a glucokinase but is indeed able to bind glucose (K(D) = 20 μM), unlike Mlc(Eco). We found that mlc of T. thermophilus is the first gene within an operon encoding an ABC transporter for glucose and mannose, including a glucose/mannose-binding protein and two permeases. malK1, encoding the cognate ATP-hydrolyzing subunit, is located elsewhere on the chromosome. The system transports glucose at 70°C with a K(m) of 0.15 μM and a V(max) of 4.22 nmol per min per ml at an optical density (OD) of 1. Mlc(Tth) negatively regulates itself and the entire glucose/mannose ABC transport system operon but not malK1, with glucose acting as an inducer. MalK1 is shared with the ABC transporter for trehalose, maltose, sucrose, and palatinose (TMSP). Mutants lacking malK1 do not transport either glucose or maltose. The TMSP transporter is also able to transport glucose with a K(m) of 1.4 μM and a V(max) of 7.6 nmol per min per ml at an OD of 1, but it does not transport mannose

The Effects of Codon Context on <i>In Vivo</i> Translation Speed

<div><p>We developed a bacterial genetic system based on translation of the <i>his</i> operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons <i>in vivo</i>. Low frequency effects of so-called “silent” codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5′- and 3′- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected <i>in vivo</i> translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated <i>in vivo</i>. This system allowed us to determine the effects of different factors on <i>in vivo</i> translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.</p></div

Histidine operon expression with His5 of the leader substituted by all 64 codons.

<p>All constructs carry a <i>hisD-lac</i> operon fusion that places the <i>lac</i> operon under control of the <i>his</i> operon promoter-attenuator regulatory system. The number in each box is the β-galactosidase activity of each mutant construct divided by the activity of the wild-type construct that contains the CAC His codon at His5. This figure also shows all tRNA species represented by a single or multiple solid black circles connected by lines according to Glenn Bjork <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004392#pgen.1004392-Bjork1" target="_blank">[61]</a>. If a tRNA reads a single codon, it is represented by a single black dot (ie. UGG Trp). If a given tRNA reads multiple codons the codons it reads are represented by solid black dots connected by lines.</p

Expression of the <i>his</i> operon for specific arginine and glycine codons at the His leader on ribosome stalling.

<p><b>A</b>. The levels of <i>hisD-lac</i> transcription for all strains with the His4-His5 codon positions in the His leader substituted with the 36 possible Arg-Arg codon pairs were determined by phenotypic assay on MacConkey-lactose (Mac-Lac) and tetrazolium-lactose (Tz-Lac) indicator medium. On Mac-Lac plates increasing levels of <i>lac</i> operon expression result in a darkening of red colony color transitioning from white to dark red while on Tz-Lac plates the reverse is true: on Tz-Lac plates, increased <i>lac</i> operon expression transitions from dark red to a white colony phenotype (see Fig. <b>7C</b>). By examining Lac morphologies in Mac-Lac and Tz-Lac indicator plates we were able to separate the His4-His5 Arg-Arg codon pair strains (labeled B–G), which are represented by the indicated color phenotypes where white represents the lowest level of <i>lac</i> operon expression; the transition from light pink to darker shades of red to dark violet at the highest level of <i>lac</i> operon expression. <b>B</b>. The effect of <i>argU</i> over-expression on <i>hisD-lac</i> transcription with each of the 36 possible Arg-Arg codon pairs placed at positions His4-His5 of the his leader peptide gene. The levels of <i>hisD-lac</i> expression is indicated by color shading (labeled B–F). A shift in <i>hisD-lac</i> expression is depicted by an arrow indicating the change in expression level from the parent strains in <b>A</b> that are lacking the <i>argU</i> over-expression plasmid. <b>C</b>. This figure indicates the color scheme used to determine that <i>hisD-lac</i> expression levels based on phenotype of thick background cells and individual colonies on Mac-Lac and Tz-Lac indicator plates. The wild-type strain with histidine codons at His4-His5 was used to indicate the expression level “A”. All of the Arg-Arg pairs at His4-His5 exhibited expression levels in the “B-G” range. The approximate β-galactosidase activity for each level is based on measured levels for the seven specific codon pairs at His4-His5 shown. <b>D</b>. The levels of <i>hisD-lac</i> transcription for strains with single glycine codons at His5 compared to (i) the wild-type all-His codons, (ii) to Gly-Gly codons GGA-GGU, GGU-GGA, GGC-GGC, GGA-GGA, GGU-GGU at His4-His5 and (iii) a perfect Shine-Dalgarno sequence at His1-His2, which includes the 5′-“G” nucleotide of a GCA Ala codon at His3. Levels of deattenuation were determined by phenotypic assay on MacConkey-lactose (Mac-Lac) and tetrazolium-lactose (Tz-Lac) indicator medium as described in Figure <b>7C</b>.</p

Codon pairs at His4-His5 that result in a Tz-Lac⁺ phenotype.

<p>*Calculated Anti-SD free energies <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004392#pgen.1004392-Li1" target="_blank">[27]</a> are shown in parentheses.</p

Histidine operon expression phenotypes of UCA-NNN and NNN-UCA substitutions at His4-His5 of the His leader peptide.

<p>The <i>his</i> operon leader peptide was altered with either NNN-UCA (3′-effect) substituted at positions His4-His5, or UCA-NNN (5′-effect) substituted at positions His4-His5. All constructs carry a <i>hisD-lac</i> operon fusion that places the <i>lac</i> operon under control of the <i>his</i> operon promoter-attenuator regulatory system. Levels of <i>hisD-lac</i> transcription are qualitative as determined by a color phenotype on MacConkey lactose indicator medium where white is Lac⁻ and the redder the Lac⁺ colonies, the greater the levels of β-galactosidase expressed from <i>hisD-lac</i>.</p