Development of Assay Systems for Amber Codon Decoding at the Steps of Initiation and Elongation in Mycobacteria (original) (raw)
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PloS one, 2016
Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis), and an aminoacyl-tRNA synthetase (AARS) mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properl...
PLOS Genetics, 2015
RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteomeprofiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.
Translational regulation in mycobacteria and its implications for pathogenicity
Nucleic acids research, 2018
Protein synthesis is a fundamental requirement of all cells for survival and replication. To date, vast numbers of genetic and biochemical studies have been performed to address the mechanisms of translation and its regulation in Escherichia coli, but only a limited number of studies have investigated these processes in other bacteria, particularly in slow growing bacteria like Mycobacterium tuberculosis, the causative agent of human tuberculosis. In this Review, we highlight important differences in the translational machinery of M. tuberculosis compared with E. coli, specifically the presence of two additional proteins and subunit stabilizing elements such as the B9 bridge. We also consider the role of leaderless translation in the ability of M. tuberculosis to establish latent infection and look at the experimental evidence that translational regulatory mechanisms operate in mycobacteria during stress adaptation, particularly focussing on differences in toxin-antitoxin systems be...
Organization and copy number of initiator tRNA genes in slow- and fast-growing mycobacteria
Journal of Biosciences, 1998
We have previously reported the isolation and characterization of a functional initiator tRNA gene, metA, and a second initiator tRNA-like sequence, metE, from Mycobacterium tuberculosis. Here we describe the fine mapping of the initiator tRNA gene locus of the avirulent (H37Ra) and virulent (H37Rv) strains of M. tuberculosis. The genomic blot analyses show that the 1.7 kb (harbouring metE) and the 6.0 kb Bamffi (harbouring metA) fragments are linked. Further, sequencing of a portion of the 6.0 kb fragment, in conjunction with the sequence of the 1.7 kb fragment confirmed the presence of an IS6110 element in the vicinity of metE. The IS element is flanked by inverted (28 bp, with 3 contiguous mismatches in the middle) and direct (3 bp) repeats considered to be the hallmarks of IS6110 integration sites. The organization of the initiator tRNA gene locus is identical in both the H37Ra and H37Rv strains and they carry a single copy of the functional initiator tRNA gene. Interestingly, the fast growing Mycobacterium smegmatis also bears a single initiator tRNA gene. This finding is significant in view of the qualitative differences in total tRNA poolsand the copy number of rRNA genes in the fast-and slow-growing mycobacteria. Finally, we discusshypotheses related to the origin of metE in M. tuberculosis.
PLoS ONE, 2013
While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the a-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA His have distinctive identity elements, we constructed E. coli tRNA His CUA , a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon-containing reporter genes (encoding a b-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA His CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA His CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA His CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair. Citation: Ko J-h, Llopis PM, Heinritz J, Jacobs-Wagner C, Sö ll D (2013) Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNA His Pair. PLoS ONE 8(12): e83630.
Effects of a Minor Isoleucyl tRNA on Heterologous Protein Translation inEscherichia coli
1995
Effects of a minor isoleucyl tRNA on http://jb.asm.org/content/177/24/7086 Updated information and services can be found at: These include: CONTENT ALERTS more» cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:
Translation initiation is a sequential process involving interactions between the 30S small ribosomal subunit, initiation factors and initiator tRNA. The Escherichia coli K-12 strain is unique in the Escherichia because it has two different initiator tRNA sequences, tRNAfMet1 encoded by the metZWVgenes and tRNAfMet2 encoded by the metY gene. A (F) tRNA fMet2 (CUA) expression results in decreased fitness in Crooks, W, and K-12 strains. All strains harbor the pULTRA::tac-metY(CUA) plasmid. Ratios on the Y-and Xaxis were calculated by dividing growth rate and max OD600 from cultures induced with 1 mM IPTG to those repressed with 2% glucose. Each data point is the average of three biological replicates. Error bars represent one standard deviation.
FEMS Microbiology Letters, 2009
Two dissimilar seryl-tRNA synthetases (SerRSs) exist in Methanosarcina barkeri: one of bacterial type (bMbSerRS) and the other resembling SerRSs present only in methanogenic archaea (mMbSerRS). While the expression of the archaeal bMbSerRS gene in Escherichia coli complements the function of thermolabile SerRS at a nonpermissive temperature, mMbSerRS does not. Our recent X-ray structural analysis of mMbSerRS revealed an idiosyncratic N-terminal domain and a catalytic zinc ion in the active site, identifying methanogenic-type SerRSs as atypical members of the SerRS family. To shed further light on substrate discrimination by methanogenic-type SerRS, we developed an in vivo system in E. coli to study tRNA serylation by mMbSerRS variants. We show that coexpression of the M. barkeri SerRS gene, encoding either bacterial- or methanogenic-type SerRS, with the gene for cognate archaeal suppressor tRNA leads to suppression of bacterial amber mutations, implying that the E. coli translation machinery can use serylated tRNA from methanogenic archaea as a substrate in protein synthesis. Furthermore, because serylation of M. barkeri serine-specific tRNA by endogenous E. coli SerRS is negligible, suppression is entirely dependent on recognition between archaeal partners (mMbSerRS/suppressor tRNA(Ser)). Thus, the efficiency of suppression by mMbSerRS variants quantified in the described beta-galactosidase-based reporter system, accurately reflects enzymes' serylation propensity obtained by in vitro kinetic measurements.