In vivo transcription of a eukaryotic regulatory gene.

Losson, Régine; Fuchs, Robert P. P.; Lacroute, François

doi:10.1002/j.1460-2075.1983.tb01720.x

Cited by 43 publications

(23 citation statements)

References 23 publications

(21 reference statements)

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“…With several bicistronic viral mRNAs, the (poly)peptide encoded in the upstream (mini)cistron has been detected (17,22,25,69), confirming that the leader sequence is indeed translated. In a few cellular mRNAs, the upstream ORF terminates very close to the ATG codon that initiates the major ORF (10,24,42,59,66); I would expect such mRNAs to reinitiate inefficiently, by analogy with the constructs described in Fig. 3.…”

Section: Discussionmentioning

confidence: 99%

“…Sherman and Stewart (62) concluded from their study of cytochrome c mutants that yeast ribosomes cannot reinitiate, but in all of their mutants the upstream minicistron terminated very close to the ATG triplet that would have been used to reinitiate cytochrome c. That motif might be unfavorable for reinitiation in yeast, as it is in higher eucaryotes. Since upstream ATG codons, invariably followed by a terminator codon, have been found in a number of yeast mRNAs (18,24,42,50,56,66,67), it seems likely that yeast ribosomes either reinitiate or undergo leaky scanning, but neither mechanism has yet been established experimentally in yeast.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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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“…Culbertson, and A. Jacobson, in prep.). PPR1 encodes an inherently unstable mRNA (Losson et al 1983), which we have shown is stabilized by loss of UPF1 function (S.W. Peltz, P. Leeds, M.R.…”

Section: Genes and Developmentmentioning

confidence: 99%

“…The leader region of the transcript contains two contiguous AUG codons, followed by a stop codon, three codons later (Losson et al 1983). Therefore, it may contain a premature stop codon that normally targets the mRNA for degradation by the UPFl-mediated pathway.…”

Section: Genes and Developmentmentioning

confidence: 99%

The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon.

Leeds¹,

Peltz²,

Jacobson³

et al. 1991

Genes & Development

447

462

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mRNA decay rates often increase when translation is terminated prematurely due to a frameshift or nonsense mutation. We have identified a yeast gene, UPF1, that codes for a trans-acting factor whose function is necessary for enhanced turnover of mRNAs containing a premature stop codon. In the absence of UPF1 function, frameshift or nonsense mutations in the HIS4 or LEU2 genes that normally cause rapid mRNA decay fail to have this effect. Instead, the mRNAs decay at rates similar to the corresponding wild-type mRNAs. The stabilization of frameshift or nonsense mRNAs observed in upfl-strains does not appear to result from enhanced readthrough of the termination signal. Loss of UPF1 function has no effect on the accumulation or stability of HIS4 + or LEU2 + mRNA, suggesting that the UPF1 product functions only in response to a premature termination signal. When we examined the accumulation and stability of other wild-type mRNAs in the presence or absence of UPF1, including MAT~I, STE3, ACT1, PGK1, PAB1, and URA3 mRNAs, only the URA3 transcript was affected. On the basis of these and other results, the UPF1 product appears to participate in a previously uncharacterized pathway leading to the degradation of a limited class of yeast transcripts. Nonsense mutations that generate a premature translational termination signal often reduce the steady-state accumulation of the corresponding mRNA (Brown 1989;Peltz et al. 1990). In a study of the yeast URA3 gene, it was shown that the extent of reduced mRNA accumulation depends on the position of the nonsense mutation (Losson and Lacroute 1979). Mutations near the 5' end of the transcript were shown to have a greater destabilizing effect than mutations near the 3' end. Furthermore, introduction of an amber tRNA suppressor restabilized ura3 nonsense mRNA, indicating that the turnover rate is determined in part by the relative efficiencies of termination versus readthrough of the stop codon. These studies suggested that the turnover rate of nonsense mRNA is probably related to some aspect of its translation rather than to a potential change in mRNA structure that might result from the presence of a nonsense mutation.Similar studies in higher eukaryotes have proven more difficult to interpret. In some cases, the introduction of a premature stop codon into a gene has been linked to 3Corresponding author. increased cytoplasmic turnover (Maquat et al. 1981;Barker and Beemon 1991). However, other studies suggest that nonsense mutations may cause changes in nuclear processing and/or transport, and these changes, rather than cytoplasmic mRNA degradation, may be primarily responsible for decreased steady-state mRNA levels (Humphries et al. 1984;Takeshita et al. 1984;Urlaub et al. 1989;Cheng et al. 1990).Here, we report the characterization of mutations in the yeast Saccharomyces cerevisiae that specifically stabilize mRNAs containing a premature translational termination signal. The mutations arose in a strain containing his4-38, a + 1 frameshift mutation near the 5' end of the HIS4 tr...

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“…A further modification of the scanning model of translation initiation states that 40s ribosomal subunits may bypass AUG codons if they do not exist in an optimal sequence context (Kozak, 1984;Losson et al, 1983). The results reported here would tend to support this model since for the A,, serotype which possesses a favourable initiation site for P20a a significant amount of this protein is synthesized.…”

Section: B E C L a R K E A N D Othersmentioning

confidence: 61%

Two Initiation Sites for Foot-and-Mouth Disease Virus Polyprotein in vivo

Clarke¹,

Sangar²,

Burroughs

et al. 1985

Journal of General Virology

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SUMMARYTypically, the translation of eukaryotic mRNAs into protein is initiated at a single site. However, we have recently shown that not one but two primary products, P20a and P I6, are translated from the 5' end of the coding region of the genome of foot-andmouth disease virus (FMDV). In this paper we show by partial protease digestion of these proteins that they differ only at their N termini, thus confirming the presence of two initiation sites for translation of FMDV RNA. Sequence analysis of two subtypes of the virus (Al0 and A12) confirms the presence of two initiator AUG codons in the expected position on the genome. By correlation with protein synthesis data from these subtypes it appears that the relative use of each initiation site is dependent on its surrounding nucleotide sequence. In addition, the ratio of the two proteins when synthesized in vitro differs markedly from that when they are synthesized in vivo, suggesting the presence of a control mechanism for synthesis of P20a in vivo which may be absent in vitro. We also show that the cleavage site between these two proteins and the structural protein precursor, P88, is located closer to the N terminus of the polyprotein than has previously been reported.

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In vivo transcription of a eukaryotic regulatory gene.

Cited by 43 publications

References 23 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 product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon.

Two Initiation Sites for Foot-and-Mouth Disease Virus Polyprotein in vivo

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