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J Biomol Tech. 2007 February; 18(1): 60.
PMCID: PMC2291868

P173-M Slippery when Translated: Extensive Programmed Ribosomal Frameshifting Revealed by Proteomics

Abstract

Living organisms translate genetic information in many ways, occasionally altering transcription signals and changing reading frames. A consequence of these post-transcriptional actions (recoding) is that a single coding sequence can produce multiple gene products, or a single product from two overlapping open reading frames (ORFs). Although examples of recoding have been uncovered in all well-studied organisms from viruses to bacteria to humans, we know little about the global contribution recoding makes to proteomic complexity. Proteomic methods tailored to expose recoding events will have enormous impact.

In examining the proteome of Syntrophus aciditrophicus, a gram-negative bacterium that grows syntrophically (cooperatively) with methanogens or sulfate reducers, we found several examples of translational frameshifting, apparently arising from particularly “slippery” mRNA sequences (XXXY YYZ). De novo sequencing of LC-MS/MS spectra obtained from trypsin-digested 2D gel spots reveals multiple peptide sequences arising from a single stretch of mRNA sequence, providing a rare opportunity to view proteins generated in vivo by recoding. Linking sequenced peptides to 2D gel spots presents the extraordinary opportunity to monitor ratios of recoding products with respect to changes in culture conditions and even whether S. aciditrophicus is cultivated syntrophically or in isolation.

Our studies revealed that predicted 4- and 16-kDa ORFs actually correspond to a 30-kDa product with at least two frameshifts (peptides identified in all 3 reading frames). More than ten tryptic peptides are observed that do not correspond to the primary sequence in any single reading frame. Instead, they define the actual recoding site.

These data extend the questions about small ORFs beyond “real or unreal?” to “small or enlarged with frame-shift?” They also promise to elucidate factors regulating frameshifts, translational bypassing, and readthrough. Finally, they raise important questions about present capabilities to predict and quantify gene products, and future capabilities needed to address these issues in high-throughput.


Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities