Localization of the target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli

Baughman, Gail; Nomura, Masayasu

doi:10.1016/0092-8674(83)90555-x

Cited by 130 publications

(83 citation statements)

References 31 publications

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“…An important distinction is that eucaryotic ribosomes seem constrained to enter only at the 5' end of the mRNA, and therefore the same 40S ribosomal subunit presumably translates the first and second cistrons. That is not necessarily true in procaryotes; the fact that the downstream cistron almost always has its own Shine-Dalgarno sequence means that bacterial ribosomes could initiate there de novo; in cases where the distal cistron is translated more efficiently than the cistron that precedes it, ribosomes must initiate de novo at the downstream site (2). The arbitrary order of binding of Met-tRNA and mRNA to bacterial ribosomes also differs from that in eucaryotes, where the small ribosomal subunit must bind Met-tRNA before the ATG initiator codon can be recognized (reviewed in reference 31).…”

Section: Discussionmentioning

confidence: 99%

“…The correlation between intercistronic length and the efficiency of reinitiation in eucaryotes is curiously just the opposite of what occurs in procaryotes. In many, albeit not all (2,47), coregulated bacterial genes, the terminator codon of one cistron overlaps the initiator codon of the next (6,9,15,23,43,49,53,72,73) and translation of the downstream cistron is drastically impaired when the intercistronic distance is expanded experimentally (Fig. 6).…”

Section: Discussionmentioning

confidence: 99%

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Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.

Kozak¹

1987

Mol. Cell. Biol.

502

433

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Simian virus 40-based plasmids that direct the synthesis of preproinsulin during short-term transfection of COS cells have been used to probe the mechanism of reinitiation by eucaryotic ribosomes. Earlier studies from several laboratories had established that the ability of ribosomes to reinitiate translation at an internal AUG codon depends on having a terminator codon in frame with the preceding AUG triplet and upstream from the intended restart site. In the present studies, the position of the upstream terminator codon relative to the preproinsulin restart site has been systematically varied. The efficiency of reinitiation progressively improved as the intercistronic sequence was lengthened. When the upstream "minicistron" terminated 79 nucleotides before the preproinsulin start site, the synthesis of proinsulin was as efficient as if there were no upstream AUG codons. A mechanism is postulated that might account for this result, which is somewhat surprising inasmuch as bacterial ribosomes reinitiate less efficiently as the intercistronic gap is widened.Eucaryotic ribosomes usually initiate at the AUG codon that lies closest to the 5' end of the mRNA (31). That tendency has been rationalized by postulating that the small ribosomal subunit binds initially at the capped 5' end of the mRNA and subsequently migrates to the AUG initiator codon, which is recognized more or less efficiently depending on nearby sequences. From a comparison of several hundred vertebrate mRNAs, GCCACCAUGG has been proposed as the consensus sequence for initiation in higher eucaryotes (30, 33, Kozak, submitted for publication). The contribution of every nucleotide in that motif has been confirmed by mutagenesis (36; Kozak, J. Mol. Biol., in press). The most highly conserved nucleotides are the purine, most often A, in position -3 (i.e., three nucleotides upstream from the AUG codon) and the G in position +4; the aforementioned mutational analyses confirmed that those two positions have the strongest influence on initiation. Thus, a potential initiator codon can usually be designated as "strong" or "weak" by considering only those positions.There are ways for eucaryotic ribosomes to initiate at internal sites in certain mRNAs, but those sites can be reached only by advancing from the 5' end; never, it seems, by direct internal binding. An early indication of the constraint against direct internal binding was the inability of eucaryotic ribosomes to bind to circular RNA templates (27,28), and a considerable body of other evidence has since been adduced (see Discussion). One mechanism by which ribosomes can reach an internal AUG codon is "leaky scanning," which occurs when the 5'-proximal AUG codon lies in an unfavorable context for initiation. Data consistent with leaky scanning have come from manipulating the sequences of synthetic constructs (35,36,40) and from analyzing naturally occurring viral mRNAs that are bifunctional (reviewed in references 38 and 39). A second mechanism for reaching an internal AUG codon operates when the upstre...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.

Kozak¹

1987

Mol. Cell. Biol.

502

433

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“…Although the mechanism of translational coupling in the trp operon did not involve mRNA binding by a translational repressor, Nomura et al reasoned that translational coupling might also occur in r-protein operons. They demonstrated that a single repressor r-protein binding event could inhibit expression of an entire r-protein operon, because translation of each cistron in the mRNA was coupled to translation of the target cistron at which the translational repressor acted (28,29). A general mechanism was envisioned in which inhibition of translation of upstream cistrons in r-protein operons resulted in the formation of mRNA secondary structures that precluded translation of the downstream cistrons.…”

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confidence: 99%

“…In the L11-L1 operon, the translational repressor L1 binds to a site on the mRNA upstream of the L11 cistron. The L1 binding site on the mRNA resembles the L1 binding site on 23S rRNA, and L1 inhibits translation of both proteins by binding to its mRNA target (29). In the L35-L20 operon, which is transcribed from several promoters (thrS, infC P1, infC P2, and rpmI), L20 (encoded by rplT) binds upstream of the L35 coding region, blocks the L35 ribosome binding site, and allows a secondary structure to form downstream that occludes the L20 ribosome binding site, blocking both L35 and L20 translation (30).…”

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confidence: 99%

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Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppGpp and DksA

Lemke

Sanchez-Vazquez²,

Burgos³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

133

144

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We show here that the promoters for many of the Escherichia coli ribosomal protein operons are regulated directly by two transcription factors, the small RNA polymerase-binding protein DksA and the nutritional stress-induced nucleotide ppGpp. ppGpp and DksA work together to inhibit transcription initiation from ribosomal protein promoters in vitro and in vivo. The degree of promoter regulation by ppGpp/DksA varies among the r-protein promoters, but some are inhibited almost as much as rRNA promoters. Thus, many r-protein operons are regulated at the level of transcription in addition to their control by the classic translational feedback systems discovered ∼30 y ago. We conclude that direct control of r-protein promoters and rRNA promoters by the same signal, ppGpp/DksA, makes a major contribution to the balanced and coordinated synthesis rates of all of the ribosomal components.ribosome synthesis | transcriptional control | stringent response | translational control P rotein synthesis is the major consumer of cellular energy in bacteria. Because the number of ribosomes is the primary determinant of the level of translation, and ribosome synthesis itself is an energy-intensive process, there are mechanisms that prevent over-or underinvestment of cellular resources in ribosome synthesis (1). Both ribosomal RNA (rRNA) and ribosomal protein (r-protein) synthesis rates are thereby tightly regulated in Escherichia coli (for reviews, see refs. 2, 3).One of the earliest reported examples of regulation of bacterial ribosome synthesis is a stress response referred to as "the stringent response," in which rRNA transcription is inhibited in cells starved for amino acids or some other nutrients (4). In this response, uncharged tRNAs induce the ribosome-associated RelA and/or SpoT proteins to synthesize ppGpp (5-7). [The term "ppGpp" is used here to describe both the unusual nucleotide guanosine-3′,5′-(bis)pyrophosphate and its pentaphosphate precursor.] ppGpp concentrations change not only after complete starvations but also after less severe shifts in nutritional conditions, coordinating rRNA synthesis with the need for protein synthesis. Shifts to a more favorable nutritional condition result in a decrease in the concentration of ppGpp and a corresponding increase in rRNA promoter activity, whereas shifts to a less favorable condition result in an increase in ppGpp and a corresponding decrease in rRNA transcription (8).ppGpp binds directly to E. coli RNA polymerase (RNAP) and inhibits transcription from rRNA promoters (9), although the identity of the ppGpp binding site on RNAP remains unclear (10). However, for ppGpp to exert its full effect on transcription, RNAP has to be modified by the small protein, DksA (11,12). Unlike ppGpp, DksA is present at high concentrations in cells under all conditions that have been examined (11,13). rRNA transcription initiation is also regulated by the concentration of the first nucleotide in the transcript (8,14) and by at least one DNA binding factor, the 11.2-kDa Fis protein (15). Tog...

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Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs.

1988

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Chloroplast genes are typically organized into polycistronic transcription units that give rise to complex sets of overlapping RNAs through a series of processing steps. The functional significance of this complicated mode of expression is unknown. To determine whether processing of the primary transcript is required to create translatable mRNAs, the translational properties of the RNAs derived from the maize psbB gene cluster (containing the psbB, psbH, petB and petD genes) were examined. Almost all of the approximately 20 RNAs derived from this region co‐sediment with polysomes in sucrose gradients, suggesting that at least one coding region on most transcripts is translated. To determine which sequences are translated on each polycistronic RNA, antibodies to psbB, petB or petD proteins were used to immunoselect polysomes engaged in the synthesis of each protein. Northern and S1 nuclease analyses of the immunoselected RNAs revealed that (i) potential start codons within the petB and petD introns are not functional in translation; (ii) all transcripts containing spliced petB or petD sequences are translated to give these proteins, regardless of upstream or downstream sequences; (iii) psbB is translated from all transcripts encoding it. It is concluded that intercistronic processing is not required for translation of these RNAs, although certain processing steps may enhance translational efficiency.

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Localization of the target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli

Cited by 130 publications

References 31 publications

Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.

Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.

Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppGpp and DksA

Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs.

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