A role for mRNA secondary structure in the control of translation initiation

Hall, Michael N.; Gabay, Joëlle E.; Débarbouillé, Michel; Schwartz, Maxime

doi:10.1038/295616a0

Cited by 335 publications

(153 citation statements)

References 25 publications

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144

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“…This conclusion is in line with other evidence which points to the degree of masking of the initiator region by RNA secondary structure as an important determinant of ribosome binding site strength (3,19,25,49). However, it is unlikely that a simple proportional relationship exists between accessibility and RBS strength, since there are other determinants such as the S-D sequence and AUG.…”

supporting

confidence: 77%

“…These results indicate that the expression of the 5PR-01 and -04 mutations is probably dependent upon neighbouring downstream lac Z coding sequences (8,25,49). DISCUSSION The selection for up mutations as described in this paper is analogous to the method of maximizing gene expression in which the structure and distance between promoter and gene is varied by limited exonuclease digestion (30).…”

Section: Dna Segregation Analysismentioning

confidence: 96%

“…Alternatively, the -01 and -04 mutations could change the secondary (or tertiary) structure of the whole ribosome binding area including the proximal part of the lac Z coding region (25,49).…”

Section: Dna Segregation Analysismentioning

confidence: 99%

“…Other investigators have postulated or provided evidence that mRNA secondary structure masking effects are important in translation initiation (3,9,19,25,49,54).…”

mentioning

confidence: 99%

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Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

Warburton¹,

Boseley²,

Porter³

1983

Nucl Acids Res

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A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E.coli trp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased a-galactosidase synthesis, were selected for DNA sequencing. One clone has a G--A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C--T and T--C) between the Shine-Dalgarno sequence and initiation codon which raise expression of a-galactosidase two-fold.A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present.These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.

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supporting

confidence: 77%

Section: Dna Segregation Analysismentioning

confidence: 96%

Section: Dna Segregation Analysismentioning

confidence: 99%

“…Other investigators have postulated or provided evidence that mRNA secondary structure masking effects are important in translation initiation (3,9,19,25,49,54).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

Warburton¹,

Boseley²,

Porter³

1983

Nucl Acids Res

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“…These nucleotide sequences (12,13,14), the exact SD-AUG spacing (15) and the three nucleotides 3' of the AUG (14) have all been shown to affect the efficiency of translation. In addition there is evidence that translation is much reduced if parts of the SD (16) or the AUG (17) are involved in RNA secondary structure. The relative contributions of the above parameters are not yet precisely defined or predictable (18).…”

Section: Introductionmentioning

confidence: 99%

Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library

Hallewell¹,

Masiarz²,

Najarian³

et al. 1985

Nucl Acids Res

143

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The molecular cloning and nucleotide sequence of the cDNA for human Cu/Zn superoxide dismutase (SOD) is reported. The tacI promoter has been used to direct the synthesis in E. coli of this SOD which is soluble, stable, and of normal specific activity. The N-terminal methionine is removed from this protein. A construction with a ribosome binding site identical to that of the lacz gene 5' of the initiator methionine codon, resulted in low levels of SOD. An in vitro mutagenesis procedure was used to randomise the four nucleotides preceding the initiator methionine codon and the silent third positions of the codons specifying the second and third amino acids. Analysis of a sample of 500 clones showed that ca. 25 clones synthesised 5% or more of soluble cell protein as SOD. The nucleotide sequences of high level expressors showed a predominance of A and T residues in t e variable positions 5' of the initiator methionine codon. An SOD mutant (ala +gln) was discovered during the sequencing and shown to lack dismutation activity. Secondary structure predictions for the 5' regions of the mRNAs from high and low level expressors support the hypothesis that initiation of translation is much reduced if part of the region complementary to 16s rRNA is base paired in a stem structure.

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