Features of a Leader Peptide Coding Region that Regulate Translation Initiation for the Anti-TRAP Protein of B. subtilis

Abstract

The rtpA gene of Bacillus subtilis encodes the Anti-TRAP protein, AT. AT can bind and inhibit the TRAP regulatory protein, preventing TRAP from promoting transcription termination in the trpEDCFBA operon leader region. AT synthesis is upregulated transcriptionally and translationally in response to the accumulation of uncharged tRNA(Trp). Here we analyze AT's translational regulation by rtpLP, a 10 residue leader peptide coding region located immediately preceding the rtpA Shine-Dalgarno sequence. Our findings suggest that, whenever the charged tRNA(Trp) level is sufficient to allow the ribosome translating rtpLP to reach its stop codon, it blocks the adjacent rtpA Shine-Dalgarno sequence, inhibiting AT synthesis. However, when there is a charged tRNA(Trp) deficiency, the translating ribosome presumably stalls at one of three adjacent rtpLP Trp codons. This stalling exposes the rtpA Shine-Dalgarno sequence, permitting AT synthesis. RNA-RNA pairing may also influence AT synthesis. Production of AT would inactivate TRAP, thereby increasing trp operon expression.

RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria

“…11, 12, 13;Chen and Yanofsky 2004). Transcriptional regulation is based on the T box RNA-based regulatory mechanism, a mechanism used in controlling expression of many genes of Gram-positive bacteria-particularly aminoacyl-tRNA synthetase genes (Henkin 2000;Grundy and Henkin 2004).…”

“…12; Sarsero et al 2000). Whenever uncharged tRNA Trp accumulates, it binds to the leader transcript of the at operon, stabilizing an antiterminator (Chen and Yanofsky 2004). This antiterminator prevents formation of the alternative, transcription terminator, hence transcription proceeds into the at operon's structural genes, including rtpA, the structural gene for the AT protein.…”

“…This sensing is performed during attempted translation of three consecutive tryptophan codons in a 10-residue leader peptide coding region, rtpLP, located in the RNA sequence immediately following the T box sequence of the at operon (Figs. 11, 13;Chen and Yanofsky 2004). The three tryptophan codons of rtpLP are located appropriately so that, whenever uncharged tRNA Trp -mediated ribosome stalling occurs at any one of these codons, it exposes the AT Shine-Dalgarno sequence, allowing efficient initiation of AT synthesis (Fig.…”

“…Organization and features of the at operon of B. subtilis. The at operon consists of a leader regulatory region, a 10-residue leader peptide coding region, rtpLP, and two structural genes, rtpA, encoding the AT protein, and ycbK, encoding what it is believed to be an efflux transport protein (Sarsero et al 2000;Chen and Yanofsky 2004;Gollnick et al 2005;Yakhnin et al 2006a,b). Transcription of the at operon is regulated by the T box mechanism, in response to the accumulation of uncharged tRNA Trp .…”

Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction

“…Translational control of at operon expression is provided by the ability of a translating ribosome to move along the mRNA region encoding a tryptophan-rich leader peptide. Such movement is controlled by the level of charged tRNA Trp (3,20).…”