title: 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.
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Scarlet S
authors.initials: SS
authors.lastname: Shell
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
authors.forename: Pascal
authors.initials: P
authors.lastname: Lapierre
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
authors.forename: Mushtaq
authors.initials: M
authors.lastname: Mir
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Chase
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Pyle
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Richa
authors.initials: R
authors.lastname: Gawande
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: Rushdy
authors.initials: R
authors.lastname: Ahmad
authors.affiliation: ThermoFisher Scientific BRIMS Center, Cambridge, Massachusetts, United States of America.
authors.forename: David A
authors.initials: DA
authors.lastname: Sarracino
authors.affiliation: Department of Computer Science, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Ioerger
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Fortune
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
authors.forename: Keith M
authors.initials: KM
authors.lastname: Derbyshire
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Wade
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.
authors.forename: Todd A
authors.initials: TA
authors.lastname: Gray
mesh_terms: D005798:Genes, Bacterial; D009161:Mycobacterium / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D017423:Sequence Analysis, RNA
chemical_list: D012333:RNA, Messenger