Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

Peabody, David S.; Subramani, Suresh; Berg, Paul

doi:10.1128/mcb.6.7.2704

Cited by 130 publications

(86 citation statements)

References 29 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%

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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%

“…A second mechanism for reaching an internal AUG codon operates when the upstream AUG codon (in a favorable context for initiation, thus ruling out leaky scanning) is followed shortly by an in-frame terminator codon. In such cases, ribosomes can initiate-or, more correctly, reinitiate-translation at the next AUG codon downstream (20,35,40,51 Fig. 1B.…”

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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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“…Most mRNAs of eukaryotes and their viruses are monocistronic but in the case of those mRNAs which are bicistronic, the ribosomes can reinitiate translation at the next AUG codon downstream (Kozak, 1984;Liu et al, 1984;Peabody et al, 1986). The efficiency of reinitiation in such cases is usually low (Kozak, 1984;Liu et al, 1984) but this progressively improves as the intercistronic sequence lengthens (Kozak, 1987).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of translation of the bicistronic mRNA encoding human papillomavirus type 16 E6-E7 genes

Tan

Gloss

Bernard

et al. 1994

Journal of General Virology

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The transforming genes E6 and E7 of human papillomavirus (HPV) type 16 and other HPV types are expressed from a bicistronic mRNA with a characteristic spacing of 3 to 6 bp between the termination codon of E6 and the initiation codon of E7. Plasmid pSP64E6E7 which contains the reading frames of both E6 and E7 was constructed in order to study the expression of both proteins in a coupled transcription/rabbit reticulocyte translation system. Both E6 and E7 proteins were expressed simultaneously. This translation could be interfered with by antisense oligonucleotides corresponding to various regions of the transcript.

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“…A growing number of recent investigations suggests that eukaryotic ribosomes have the capacity to reinitiate translation at a downstream (internal) AUG, once they have terminated translation of an upstream open reading frame [1][2][3]. The hypothesis for translation initiation in eukaryotes [4][5][6] states that the 40 S ribosomal subunit first binds at the 5'-capped end of the mRNA.…”

Section: Introductionmentioning

confidence: 99%

“…In experimentally conceived mRNAs [1][2][3]9,10] it has been demonstrated that initiation at a downstream AUG can be severely impaired if this AUG is preceded by a favourable upstream AUG. If translation from the 5'-proximal AUG continues beyond the downstream AUG, translation from the downstream AUG can be totally abolished.…”

Section: Introductionmentioning

confidence: 99%

Expression of human pancreatic polypeptide precursors from a dicistronic mRNA in mammalian cells

Boel¹,

Berkner²,

Nexø³

et al. 1987

FEBS Letters

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The ability of eukaryotic ribosomes to reinitiate translation at downstream AUG codons on polycistronic mRNAs was used to select transfected CHO clones that secreted a precursor to the human pancreatic polypeptide (PP). In the in vitro constructed transcription unit, a viral promoter directed the synthesis of a dicistronic mRNA. The PP cDNA was placed in the Y-part of this transcript, while a DHFR cDNA was placed 3' to the PP. This dicistronic expression unit was transfected into CHO cells, and methotrexate-resistant colonies were selected. RNA-blots verified that the PP precursor and DHFR were expressed from the same dicistronic mRNA. The CHO cells synthesized the hormone precursor and secreted it through the constitutive secretory pathway.

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Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

Cited by 130 publications

References 29 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.

Mechanism of translation of the bicistronic mRNA encoding human papillomavirus type 16 E6-E7 genes

Expression of human pancreatic polypeptide precursors from a dicistronic mRNA in mammalian cells

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