Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome

Meydan, Sezen; Marks, James; Klepacki, Dorota; Sharma, Virag; Baranov, Pavel V.; Firth, Andrew E.; Margus, Tõnu; Kefi, Amira; Vázquez‐Laslop, Nora; Mankin, Alexander S.

doi:10.1016/j.molcel.2019.02.017

Cited by 139 publications

(173 citation statements)

References 70 publications

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“…Identification of novel leadered ORFs from conventional ribosome profiling data is challenging due to (i) the potential for artifactual signal in 5’ UTRs due to the binding of RNA-binding proteins ( 14 ), and (ii) masking of signal by overlapping ORFs on the same strand. Identification of novel ORFs can be facilitated by performing ribosome profiling on cells that have been treated with an antibiotic that traps initiating ribosomes, allowing elongating ribosomes to release their mRNAs ( 15–20 ). Two recent studies highlight the use of the antibiotics retapamulin and Onc112 for application of this approach to Escherichia coli ( 18, 20 ).…”

Section: Resultsmentioning

confidence: 99%

“…Identification of novel ORFs can be facilitated by performing ribosome profiling on cells that have been treated with an antibiotic that traps initiating ribosomes, allowing elongating ribosomes to release their mRNAs ( 15–20 ). Two recent studies highlight the use of the antibiotics retapamulin and Onc112 for application of this approach to Escherichia coli ( 18, 20 ). We performed a similar ribosome profiling of M. tuberculosis treated with retapamulin (“Ribo-RET” ( 20 )).…”

Section: Resultsmentioning

confidence: 99%

“…Two recent studies highlight the use of the antibiotics retapamulin and Onc112 for application of this approach to Escherichia coli ( 18, 20 ). We performed a similar ribosome profiling of M. tuberculosis treated with retapamulin (“Ribo-RET” ( 20 )). We aligned the ribosome-protected RNA fragment sequences to the M. tuberculosis genome and identified “Enriched Ribosome Footprints” (ERFs), sites of ribosome occupancy that exceed the local background (Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…A recent study used Ribo-RET to identify novel and isoform ORFs in E. coli ( 20 ). Although Ribo-RET has been applied to only two bacterial species to date, the abundance of novel and isoform ORFs in two distantly related species suggests that many bacterial transcriptomes are pervasively translated.…”

Section: Resultsmentioning

confidence: 99%

“…Due to the short length and low translation initiation levels of proteins generated by pervasive translation (Fig. 3, S5, S8) ( 18, 20 ), they likely represent a small fraction of total cellular translation. Moreover, our analysis of G/C content within codons strongly suggests that most of the proteins themselves are unlikely to be functional in M. tuberculosis (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Pervasive Translation in Mycobacterium tuberculosis

Smith

Canestrari

Wang

et al. 2019

Preprint

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ORF boundaries in bacterial genomes have largely been drawn by gene prediction algorithms. These algorithms often fail to predict ORFs with non-canonical features. Recent developments in genome-scale mapping of translation have facilitated the empirical identification of ORFs. Here, we use ribosome profiling approaches to map initiating and elongating ribosomes in Mycobacterium tuberculosis. Thus, we identify over 1,000 novel ORFs, revealing that much of the genome encodes proteins in overlapping reading frames, and/or on both strands. Most of the novel ORFs are short (sORFs), impeding their identification by traditional methods. The strong codon bias that characterizes annotated mycobacterial ORFs is not evident in the aggregate novel sORFs; hence most are unlikely to encode functional proteins. Our data suggest that bacterial transcriptomes are subject to pervasive translation. We speculate that the inefficiency of expressing spurious sORFs may be offset by positive contributions to M. tuberculosis biology through activities of a small subset.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 3 more Smart Citations

Pervasive Translation in Mycobacterium tuberculosis

Smith

Canestrari

Wang

et al. 2019

Preprint

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ABC‐F translation factors: from antibiotic resistance to immune response

et al. 2020

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Energy-dependent Translational Throttle A (EttA) from E. coli is a paradigmatic ABC-F protein that controls the first step in polypeptide elongation on the ribosome according to the cellular energy status. Biochemical and structural studies have established that ABC-F proteins generally function as translation factors that modulate the conformation of the peptidyl transferase center upon binding to the ribosomal tRNA exit site. These factors, present in both prokaryotes and eukaryotes but not in archaea, use related molecular mechanisms to modulate protein synthesis for heterogenous purposes, ranging from antibiotic resistance and rescue of stalled ribosomes to modulation of the mammalian immune response. Here we review the canonical studies characterizing the phylogeny, regulation, ribosome interactions, and mechanisms of action of the bacterial ABC-F proteins, and discuss the implications of these studies for the molecular function of eukaryotic ABC-F proteins, including the three human family members.

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Rapid Biophysical Characterization and NMR Spectroscopy Structural Analysis of Small Proteins from Bacteria and Archaea

et al. 2020

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Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution‐state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.

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Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome

Cited by 139 publications

References 70 publications

Pervasive Translation in Mycobacterium tuberculosis

Pervasive Translation in Mycobacterium tuberculosis

ABC‐F translation factors: from antibiotic resistance to immune response

Rapid Biophysical Characterization and NMR Spectroscopy Structural Analysis of Small Proteins from Bacteria and Archaea

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