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Feedback regulation of rRNA and tRNA synthesis and …

Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA.

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ppGpp is the down regulation of the rRNA and tRNA transcription ..

Other features of the DNA, RNA, and proteins of B. subtilis are known to contribute to the regulatory events that fine tune tryptophan biosynthesis. Thus, although E. coli and B. subtilis both recognize tryptophan and uncharged tRNATrp as signals in regulating their respective trp operon, the mechanisms they use to sense these signals are quite different (). This realization supports the presumption stated at the beginning of this article—namely, that during evolution, numerous regulatory mechanisms were undoubtedly developed and tested, and some retained, depending on their effectiveness in allowing each organism to respond to its specific metabolic needs.

Control of rRNA and tRNA synthesis in Escherichia coli by guanosine tetraphosphate.

Our studies have been focused on determining the mechanism of tryptophan induction of expression of the tryptophanase (tna) degradative operon of E. coli. We have shown that the leader transcript of this operon contains three segments essential for regulation of tna operon expression: a coding sequence for a 24-residue leader peptide, TnaC; a RNA rut binding site for the Rho termination factor; and RNA pause sites required for Rho-dependent transcription termination (; ; ; ; ; , , ). These sites all precede the two structural genes of the operon, tnaA, encoding tryptophanase, the degradative enzyme, and tnaB, encoding a tryptophan permease. The tna operon leader peptide, TnaC, has been shown to regulate Rho action by instructing the translating ribosome to bind tryptophan, inhibiting TnaC–tRNAPro cleavage (; , ). Inhibition of peptidyl-tRNA cleavage causes the ribosome translating tnaC to stall at the tnaC stop codon. This stalled ribosome blocks the Rho Factor binding site—the rut site—preventing the Rho factor from binding to the transcript and terminating transcription (). When Rho cannot bind and act, the polymerase stalled downstream in the leader region resumes transcription, proceeding into the structural genes of the operon (). If cells do not contain inducing levels of tryptophan, the ribosome translating tnaC completes TnaC synthesis, the TnaC–peptidyl–tRNA is cleaved, and the translating ribosome is released. The rut site in leader RNA is then free to bind the Rho factor, allowing Rho to bind and contact the polymerase paused in the operon's leader region, terminating transcription. E. coli's tna operon regulatory region resembles the tna operon regulatory region of several other bacterial species.

Regulation of RNA after synthesis - Encyclopedia Britannica

The at operon's leader region is designed to sense the accumulation of uncharged tRNATrp both transcriptionally and translationally, in regulating synthesis of the AT protein (, , ; ). 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 (; ). The transcript of the at operon's leader region forms a typical T box structure, with this T box RNA designed to bind and respond to uncharged tRNATrp (; ). Whenever uncharged tRNATrp accumulates, it binds to the leader transcript of the at operon, stabilizing an antiterminator (). 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. Charged tRNATrp availability is also sensed translationally, by other features of the at operon's leader transcript. 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 (, ; ). The three tryptophan codons of rtpLP are located appropriately so that, whenever uncharged tRNATrp-mediated ribosome stalling occurs at any one of these codons, it exposes the AT Shine–Dalgarno sequence, allowing efficient initiation of AT synthesis (). However, when there is sufficient charged tRNATrp for the translating ribosome to complete synthesis of the at operon's leader peptide, this ribosome would reach the rtpLP stop codon, where it would block the rtpA Shine–Dalgarno sequence. This would inhibit initiation of AT synthesis, until the ribosome is released (). We suspect that ribosome release at this leader peptide stop codon is slow, since synthesis of AT is poor whenever cells have moderate levels of charged tRNATrp. The alternative events involved in expression of the at operon are summarized in .

The stages in tryptophan and TRAP regulation of expression of the trp suboperon are summarized in . Transcription pausing (, Stage 1) and TRAP's ability to disrupt or prevent formation of the antiterminator (, Stage 2a) are the crucial events that permit this tryptophan-activated protein to regulate transcription of the trp suboperon of the aro supraoperon (). But other regulatory options exist here as well. It is important to the organism to be able to shut down translation of trpE mRNA whenever tryptophan is present in excess. Thus, TRAP-induced formation of the terminator structure is only sufficient to cause about 90%–95% transcription termination. In the 5%–10% of TRAP-bound transcripts that have not terminated, a hairpin structure forms that prevents initiation of translation of trpE. This structure reduces synthesis of anthranilate synthase, the enzyme catalyzing the first reaction of the tryptophan biosynthetic pathway. This inhibition spares chorismate for other purposes. Transcriptional pausing has been shown to be instrumental in facilitating this translation inhibition (,). This second mechanism further reduces the overall rate of tryptophan synthesis (). As we shall see, TRAP's ability to function is also influenced by a second protein, AT. The level of AT formed by cells is dependent on their levels of uncharged versus charged tRNATrp ().

Fine Regulation of RNA Synthesis

The availability of tryptophan-charged tRNATrp is also sensed as a regulatory signal in controlling trp operon transcription—by a transcription attenuation mechanism. The relevant features of the ~160 nucleotide (nt) trp operon leader transcript responsible for charged tRNATrp sensing, and for regulation by transcription attenuation, are shown in (; ). Transcription attenuation mechanisms generally involve several sequential stages or events. The stages used in attenuation regulation of the trp operon of E. coli are described in (; ). An essential feature of this attenuation mechanism is the synchronization of translation of a 14-residue leader peptide coding region, trpL, with transcription of the operon's leader region. Synchronization is achieved by exploiting features of the initial segment of the leader transcript, the segment overlapping trpL. This segment can form an RNA hairpin structure, designated hairpin 1–2, called the anti-antiterminator. Hairpin 1:2 also serves as a transcription pause signal (see , ). Transcriptional pausing is relieved when a ribosome binds at the trpL mRNA start codon and initiates synthesis of the TrpL leader peptide. The moving ribosome appears to disrupt the RNA pause hairpin, releasing the paused RNA polymerase (, Stage 1). Subsequently, either of two events occurs, depending on the availability of uncharged versus charged tRNATrp. When most of the cellular tRNATrp is uncharged, difficulty in translating the two Trp codons of trpL mRNA results in ribosome stalling at one of these Trp codons (, , Stage 2b). This allows the antiterminator structure, hairpin 2–3, to form (), which prevents formation of the terminator structure. Prevention of terminator formation allows transcription to continue into the structural genes of the operon (, Stage 2b). When charged tRNATrp is plentiful, however, translation of trpL is completed, and the translating ribosome dissociates at the trpL stop codon. This permits the leader transcript to fold and form the anti-antiterminator and terminator structures, 1:2 and 3:4 (), promoting transcription termination (, Stage 2a). Thus, depending on the availability of charged tRNATrp during translation of trpL, transcription of the structural gene region of the trp operon will or will not proceed. Many amino acid biosynthetic operons of Gram-negative bacterial species are regulated by similar ribosome-mediated transcription attenuation mechanisms (). The unique distinguishing feature of each is the inclusion of codons for the respective amino acid in the corresponding leader peptide coding region.

We have constructed a conditional rRNA gene expression system by fusing a plasmid-encoded rrnB operon to the lambda PL promoter/operator. It was thereby possible to study the events that lead to the regulation of chromosomal rRNA and tRNA synthesis after overproduction of rRNA. rRNA induction resulted in a 2-fold increase in 30S and 50S free ribosomal subunits, whereas the polysome fraction was unaffected. Overproduction of rRNA and "free" ribosomes produced a large repression of rRNA and tRNA synthesis from chromosomal genes and a smaller increase in the concentration of guanosine tetraphosphate. These results lend support to the ribosome feedback regulation model: rRNA and tRNA operons are negatively regulated, either directly or through some intermediate, by free, nontranslating ribosomes.

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  • 31/08/2011 · Genetics Research International is a ..

    Regulation of the protein synthesizing machinery-ribosomes, tRNA, factors, and so on.

  • which transcribes 5S rRNA, tRNA, ..

    Helmholtz Zentrum München - German Research Center for Environmental Health - Annual Report 2011

  • 01/02/1985 · National Academy of Sciences ..

    Vol 4 Issue 6

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Translational Control, Protein Synthesis, and RNA Regulation

Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nus B5 mutant of Escherichia coli.

like coding, decoding, regulation, ..

Overproductionof rRNA and "free" ribosomes produced a large repression of rRNA andtRNA synthesis from chromosomal genes and a smaller increase in theconcentration of guanosine tetraphosphate.

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