A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair

Das, Abhimanyu; Yanofsky, Charles

doi:10.1093/nar/12.11.4757

Cited by 112 publications

(69 citation statements)

References 37 publications

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“…Recent experiments from other laboratories (51,68) (i) With polycistronic procaryotic transcripts, where occlusion can indeed occur, the inhibitory effect of an overlapping upstream cistron is sometimes only two-or threefold (11,19,61). In eucaryotes, on the other hand, initiation at a downstream ATG codon is completely suppressed when it is overlapped by an upstream cistron (8,35,40,54,65).…”

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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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%

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

Kozak¹

1987

Mol. Cell. Biol.

502

433

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“…Mutational analysis showed that repA is translationally coupled to repB, a gene encoding a small leader peptide, and that there is almost no expression of repA in the absence of expression of repB. The stop codon of repB overlaps the repA start codon, an arrangement that is generally considered to be most efficient for coupled genes since the ribosome terminating translation of the upstream gene can reinitiate at the downstream one without dissociating from the mRNA (2,8,33,41,48). However, this was found not to be the case for the IncB plasmid pMU720, whose coupled genes, repB and repA, overlap by 10 bp.…”

Section: Discussionmentioning

confidence: 99%

The replication of an IncL/M plasmid is subject to antisense control

1995

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A 2,385-bp sequence that contains the information for the autonomous replication of the IncL/M plasmid pMU604 was characterized. Genetic analyses revealed that the replicon specifies at least four structural genes, designated repA, repB, repC, and rnaI. The repA gene encodes a protein with a molecular weight of 40,861 which probably functions as an initiator for replication. The functions of the proteins of the repB and repC genes are unclear; however, mutations in the start codon of repB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled. The rnaI gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incompatibility phenotype, thus implicating its role as the main copy number determinant. RNAI exerts its effect in trans to repress the expression of repA at the posttranscriptional level. Furthermore, two complementary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA. Base-pairing between the two complementary sequences was shown to be critical for efficient repA expression. A model for the regulation of pMU604 replication involving both translational coupling and pseudoknot formation is proposed.Plasmids that employ a system of copy number control that involves inhibitor molecules binding to target sequences generally regulate their replication via a small antisense RNA molecule. The target of this inhibitory RNA is the complementary region of a large RNA transcript, whose product is essential for replication. Both antisense and target RNAs are highly structured molecules capable of assuming stable stem-andloop configurations. These structural motifs are just as important as their primary sequences for efficient and specific interaction between antisense and target molecules (6,10,32,34,43,47,52). Because the small RNAs act in trans, they are also able to affect the replication of other plasmids showing the same specificity of replication control. The ability to interact with the replication machinery of other plasmids means that antisense RNAs act as incompatibility determinants (29).Although a number of plasmids use antisense RNA to regulate the frequency with which they initiate replication, the particular molecular mechanism differs for each system. For example, replication of the ColE1 plasmids is regulated by an antisense RNA, which by binding to a preprimer RNA alters its folding and prevents its subsequent processing to form a primer for the initiation of DNA synthesis (19,24,49,50). The antisense RNAs of the pT181, FII, IncI 1 and IncB plasmids inhibit the expression of the genes coding for the essential replication initiation proteins (Rep) (13,30,31,35,42,46). In pT181, binding of the antisense alters the folding of the rep mRNA, resulting in transcriptional attenuation of the message (31). Control of copy number in the FII plasmids R1 and NR1 involves the hybridization of the antisense RNA with its complementary sequ...

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“…The expected fusion protein products for each construct are indicated to the right. change in 32 stability during the heat-shock response, we constructed a reporter system by using translational coupling that occurs at the trpB-trpA junction, in which translation of the downstream gene (trpA) depends largely on complete translation of upstream trpB (36). The entire rpoH gene including promoters but omitting the stop codon (UAA) was fused with the 92-bp trpBA junction and with lacZ (Fig.…”

Section: Methodsmentioning

confidence: 99%

Dynamic interplay between antagonistic pathways controlling the σ ³² level in Escherichia coli

Morita

Kanemori²,

Yanagi³

et al. 2000

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

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The heat-shock response in Escherichia coli depends primarily on the transient increase in the cellular level of heat-shock sigma factor 32 encoded by the rpoH gene, which results from both enhanced synthesis and transient stabilization of normally unstable 32 . Heat-induced synthesis of 32 was previously shown to occur at the translation level by melting the mRNA secondary structure formed within the 5 coding sequence of rpoH including the translation initiation region. The subsequent decrease in the 32 level during the adaptation phase has been thought to involve both shutoff of synthesis (translation) and destabilization of 32 -mediated by the DnaK-DnaJ chaperones, although direct evidence for translational repression was lacking. We now show that the heat-induced synthesis of 32 does not shut off at the translation level by using a reporter system involving translational coupling. Furthermore, the apparent shutoff was not observed when the synthesis rate was determined by a very short pulse labeling (15 s). Examination of 32 stability at 10 min after shift from 30 to 42°C revealed more extreme instability (t1/2‫02؍‬ s) than had previously been thought. Thus, the dynamic change in 32 stability during the heat-shock response largely accounts for the apparent shutoff of 32 synthesis observed with a longer pulse. These results suggest a mechanism for maintaining the intricate balance between the antagonistic pathways: the rpoH translation as determined primarily by ambient temperature and the turnover of 32 as modulated by the chaperone (and presumably protease)-mediated autogenous control.M ost organisms respond to heat or other stresses by transiently inducing molecular chaperones and other heatshock proteins (HSPs) to cope with stress-induced protein damage (1). When Escherichia coli cells are exposed to modest heat shock by a shift from 30 to 42°C, the synthesis of HSP increases for several minutes (induction phase) and gradually decreases (adaptation phase) to reach a new steady-state level within 20-30 min. The induction results from a rapid but transient increase in the cellular level of 32 (the rpoH gene product) which directs RNA polymerase to transcribe specifically from heat-shock promoters (2-4). The increase in 32 level results from both increased synthesis and stabilization of normally unstable 32 (t 1/2 ϭ 1 min), whereas the subsequent decrease in 32 has been thought to depend on shutoff of synthesis and destabilization of 32 by the DnaK-DnaJ chaperone-mediated autogenous negative control (5-9). The free pool of DnaK and͞or DnaJ was thought to act as a cellular thermometer that modulates expression of all HSPs by monitoring the state of protein folding (9-11).Heat-induced stabilization of 32 occurs rapidly although transiently. The half-life of 32 increases from 1 to 8 min for the first several minutes, and returns to about 1 min by 10 min after shift (7-9). The initial stabilization probably results from sequestering 32 away from the DnaK-DnaJ chaperones because of heat-induced accumulation of un...

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A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair

Cited by 112 publications

References 37 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.

The replication of an IncL/M plasmid is subject to antisense control

Dynamic interplay between antagonistic pathways controlling the σ ³² level in Escherichia coli

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A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair

Cited by 112 publications

References 37 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.

The replication of an IncL/M plasmid is subject to antisense control

Dynamic interplay between antagonistic pathways controlling the σ 32 level in Escherichia coli

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Dynamic interplay between antagonistic pathways controlling the σ ³² level in Escherichia coli