Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

Abstract

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 proteome-profiling 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.

Defining the Functionally Important Domain and Amino Acid Residues in Mycobacterium tuberculosis Integration Host Factor for Genome Stability, DNA Binding, and Integrative Recombination

“…However, it remained possible that mIHF purified from E. coli cells as a 25-kDa polypeptide; a longer form of the ihf gene was cloned, and the actual molecular mass of mIHF in mycobacterial cells may not be 25 kDa and lacks the N-terminal 86 residues. Moreover, RiboSeq data support the idea that IHF is translated from a downstream translation initiation site in M. smegmatis and M. tuberculosis strains (48).…”

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

“…Moreover, recent findings that prokaryotes in general have high fractions of translated leaderless mRNAs [18,19] and sORFs (small open reading frames) [20,21] make the realization of intraRNAs coding potential worth investigating. Pervasive translation of intraRNAs would include prokaryotes into the meaningfulness of protein isoforms debate [22,23].…”

Mutation in an Unannotated Protein Confers Carbapenem Resistance in Mycobacterium tuberculosis

“…1a). A recent study (20) focusing on previously unidentified transcripts and small proteins reported a transcription start site 164 bases upstream of this ORF, providing additional evidence for the existence of a previously unidentified gene. We refer to this ORF as carbapenem resistance factor A (crfA).…”