Secondary structure of mRNA and efficiency of translation initiation

Iserentant, Dirk; Fiers, Walter

doi:10.1016/0378-1119(80)90163-8

Cited by 204 publications

(70 citation statements)

References 11 publications

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“…In the experiments described here, we changed the codon usage itself as well as the mRNA sequence. Structural features within the PGK coding sequence may be relevant for the stability of the mRNA and the translation process (19,34); the silent substitutions in the coding sequence will most likely disturb this structure and therefore could affect PGK expression.…”

Section: Resultsmentioning

confidence: 99%

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

Hoekema¹,

Kastelein²,

Vasser³

et al. 1987

Mol. Cell. Biol.

175

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The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGKI gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.Although the genetic code is degenerate for most amino acids, the choice of synonymous codons in both procaryotic and eucaryotic genes is far from random (9)(10)(11)17). The nonrandom codon usage patterns in the organisms examined show two prevailing characteristics: a strong positive correlation between the gene expression level and the degree of biased codon usage, and a similar relationship between codon usage of the highly expressed genes and the relative abundance of the corresponding tRNAs (17, 18; for recent reviews see references 4 and lla).In the yeast Saccharomyces cerevisiae, more than 96% of the amino acids in highly expressed genes are encoded by a set of 25 codons (2). The genes for all abundant proteins (e.g., glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase [PGK], and alcohol dehydrogenase) use these major codons almost exclusively throughout their sequences; codons recognized by minor tRNAs are avoided (reviewed in reference 4).In this report the function of biased codon usage in the highly expressed gene for PGK (PGKJ) was studied. PGKI coding sequence with a heterologous gene, the expression level decreases by one or two orders of magnitude. It has been shown that this dramatic difference in all cases correlates with a reduced steady-state mRNA level (3,27). These observations show that in addition to the regulatory elements flanking the structural gene, the coding sequence itself contains crucial information with regard to the expression level of this gene. The choice of codons could be one of the parameters affecting expr...

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Section: Resultsmentioning

confidence: 99%

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

Hoekema¹,

Kastelein²,

Vasser³

et al. 1987

Mol. Cell. Biol.

175

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“…Inhibition of translation has been attributed to secondary structures in the initiation region of mRNAs of normal RNA bacteriophages (11,30), mutant forms of the lamB (3, 21) and cat (20) genes in Escherichia coli, and fused genes in E. coli plasmids (37). Although secondary structures in the initiation region diminish translation, there appears to be no consistent relationship between the inhibitory effect on translation and the specific sequences masked by the secondary structure, such as the AUG initiator codon and Shine-Dalgarno sequences (2,4,5,19,37). Additional experiments performed in vivo and in vitro suggest that secondary structures in eucaryotic mRNA retard translation by interfering with initiation of protein synthesis (6,12,15).…”

Section: Discussionmentioning

confidence: 99%

A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c.

Baim¹,

Pietras²,

Eustice³

et al. 1985

Mol. Cell. Biol.

138

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The CYCI-239-0 mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu-replacement of the normal -Ala-Gly-sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-l-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYCI+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYCI-239-0 mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYCI mRNA diminishes translation by inhibiting elongation.During the past 20 years, the CYCI gene and its gene product iso-l-cytochrome c in the yeast Saccharomyces cerevisiae have been the subject of numerous studies. Mutationally altered forms of at least partially functional iso-icytochromes c have been obtained from over 500 intragenic revertants of various cycl mutants. The vast majority of these altered iso-1-cytochromes c are found at a level corresponding to the level of normal iso-l-cytochrome c in wild-type strains. Thus, most changes of the amino acid sequence do not appreciably affect the level of the protein.One type of exception is the revertants that contain AUG initiator codons situated at certain abnormal positions (25, 26, 33; S. B. Baim, Ph.D. Thesis, University of Rochester, Rochester, N.Y., 1984; S. Baim and F. Sherman, unpublished data). In this investigation we describe another type of exception, the cycl-239 revertants which contain only 20o of the normal level of iso-1-cytochrome c and contain a sequence which can accommodate the formation of a hairpin structure within the translated region of the mRNA.MATERIALS AND METHODS Genetic analysis. The symbol CYCI+ or CYCI denotes the wild-type structural gene that encodes iso-l-cytochrome c. Mutations of this gene that cause a deficiency in either the amount or activity are designated by the symbol cycl followed by the allele number, e.g., cycl-17, cycl-239, etc. Intragenic revertants of specific cycl mutants in which the amount or activity of iso-l-cytochrome c has been at least partially restored are designated by the gene symbol CYCI followed by the allele number of the mutation from which they were derived and by capital letters to designate independent revertants. For example, CYCI-239-A, CYCI-239-B, etc., denote intragenic revertants of cycl-239.Conventional Zaret and Sherman (38,39). Briefly, logarithmic-phase cells from an overnight culture were inoculated at various densities into YPD (1% yeast extract [Difco], 2% Bacto-Peptone, and 2% glucose) medium. The cultures were shaken vigorously at 30°C for 18 to 24 hours. The cells were harvested at the appropriate densities and disrupted with glass beads. The lysate was adjusted to 1% sodium dodecyl sulfate and extracted several times with phenol-chloroform-isoamyl alcohol (25:24...

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“…The relative contributions of the above parameters are not yet precisely defined or predictable (18). However, the available evidence indicates that both nucleotide sequence and RNA secondary structure in the region of the initator AUG may have the greatest influence on the efficiency of translation initiation (14,19). Thus, we describe here the construction and characterisation of an SOD expression library, produced by in vitro mutagenesis, in which nucleotides have been inserted randomly in the four positions 5' of the AUG and in the silent third positions of the codons specifying the second and third amino acids.…”

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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Secondary structure of mRNA and efficiency of translation initiation

Cited by 204 publications

References 11 publications

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c.

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

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