Identification of the SV40 agnogene product: a DNA binding protein

Jay, Gilbert; Nomura, Shigeko; Anderson, Carl W.; Khoury, George

doi:10.1038/291346a0

Cited by 169 publications

(111 citation statements)

References 17 publications

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“…Because the motif is not widespread, however (nearly half of the mRNAs that are suspected to support reinitiation are derived from protooncogenes [38]), reinitiation in eucaryotic cells at large might be less efficient than in cultured COS cells. 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.…”

Section: Discussionmentioning

confidence: 88%

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: 88%

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

Kozak¹

1987

Mol. Cell. Biol.

502

433

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“…We therefore suggest that translation of the wildtype src message produces a nine-amino acid polypeptide in addition to the 60,000-dalton src protein. There are several examples of single mRNAs that appear to have more than one functional site of initiation (2,16,21,22), and two recent reports support the idea that termination codons can have a profound influence on initiation (19a, 20a).…”

Section: Resultsmentioning

confidence: 99%

Mutation of a termination codon affects src initiation.

Hughes¹,

Mellström²,

Kosik³

et al. 1984

Mol. Cell. Biol.

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The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a fuli-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src ranslation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.Rous sarcoma virus (RSV) is apparently transcribed into a single RNA species beginning at the left end of the R sequence in the left-hand long terminal repeat and terminating at the right end of the R sequence in the right-hand long terminal repeat. This transcript constitutes genomic RNA and serves as the precursor for RSV messenger RNAs. Four messenger RNAs are known: gag mRNA, which is apparently unspliced and is thought to be identical to genomic RNA; gag-pol mRNA, which may have a small region near the gag-pol junction removed by RNA splicing; env mRNA, which has most of gag and pol removed by splicing; and src mRNA, which has most of gag, pol, and env removed by splicing (30; Fig.

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“…However, the translational start does not contain the consensus purine at the -3 position. Although polycistronic messages are common in procaryotes, in eucaryotes they are known only in viral systems (3,8,15,19 (47). However, blot analysis with Antp probes of RNA from embryonic, larval, and pupal stages demonstrated the activity of both promoters at each stage ( Fig.…”

Section: Discussionmentioning

confidence: 99%