Pervasive translation in Mycobacterium tuberculosis

Abstract

Most bacterial ORFs are identified by automated prediction algorithms. However, these algorithms often fail to identify ORFs lacking canonical features such as a length of >50 codons or the presence of an upstream Shine-Dalgarno sequence. Here, we use ribosome profiling approaches to identify actively translated ORFs in Mycobacterium tuberculosis. Most of the ORFs we identify have not been previously described, indicating that the M. tuberculosis transcriptome is pervasively translated. The newly described ORFs are predominantly short, with many encoding proteins of ≤50 amino acids. Codon usage of the newly discovered ORFs suggests that most have not been subject to purifying selection, and hence are unlikely to contribute to cell fitness. Nevertheless, we identify 90 new ORFs (median length of 52 codons) that bear the hallmarks of purifying selection. Thus, our data suggest that pervasive translation of short ORFs in Mycobacterium tuberculosis serves as a rich source for the evolution of new functional proteins.

Conditional termination of transcription is shaped by Rho and translated uORFS inMycobacterium tuberculosis

“…Our results further suggested that many of the identified leaders were coding, in line with previous findings (35,36), and indicating that conditional termination of transcription is in many cases linked to translation. While we did find some commonality between our proposed leader peptides and those recently reported by Smith et al (36) (i.e. 9 exact matches and 28 isoforms, Supplementary Table 7), it appears from the number of peptides that did not match, that the methods of Ribo-seq/Ribo-ret and Term-seq are complementary in identifying potentially translated, and perhaps in particular, regulatory uORFs.…”

“…While we did find some commonality between our proposed leader peptides and those recently reported by Smith et al . (36) (i.e. 9 exact matches and 28 isoforms, Supplementary Table 7), it appears from the number of peptides that did not match, that the methods of Ribo-seq/Ribo-ret and Term-seq are complementary in identifying potentially translated, and perhaps in particular, regulatory uORFs.…”

A Practical Guide to Small Protein Discovery and Characterization Using Mass Spectrometry

“…To minimize the identification of so-called “one-hit wonders,” several PSMs (independent observations of the same peptide sequence) should be required. As small proteins are often of lower abundance ( 81 , 93 ) and hence are expected to produce few MS-detectable peptides, researchers have analyzed multiple biological conditions to increase their odds of identification, and used proteases other than trypsin ( Table 1 ) that can add more spectral evidence to support novel small protein identification ( 12 , 35 , 60 ). It is important to note that the number of false positive identifications will also increase as the size of the MS data set increases ( 94 , 95 ).…”

“…( 105 ). Ribo-seq data can also complement MS-based evidence ( 104 ), and are particularly well-suited for identification of protein start sites ( 6 , 93 , 106 ), which is more challenging with MS-based approaches. Fifteen out of 16 novel small proteins jointly implied by Ribo-seq data and predicted by sPepFinder ( 107 ) in Salmonella enterica serovar Typhimurium were validated by MS, highlighting the complementarity of MS and Ribo-seq as methods to identify small proteins ( 104 ).…”

“…There is increasing evidence that some, perhaps many, small proteins that can be detected by transcriptomic or MS-based approaches are nonfunctional, as defined by the lack of an effect on cell fitness ( 6 , 93 , 113 , 114 ). Hence, prioritization of novel small proteins for further study is a critical step ( Fig.…”