Cleavage within an RNase III site can control mRNA stability and protein synthesis<i>in vivo</i>

Panayotatos, Nikos; Kim, Truong

doi:10.1093/nar/13.7.2227

Cited by 68 publications

(37 citation statements)

References 21 publications

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“…The two mRNAs produced by cleavage at these sites are identical except for the site for inserting codons into the test gene and a 45-codon deletion in the control gene, to allow the two mRNAs (and proteins) to be separated by gel electrophoresis. Independent translation of test and control mRNAs avoids possible interactions between two coding sequences in the same mRNA, and RNase III cleavage leaves a strong stem-loop structure at the 3' end, which should stabilize the mRNAs (6,11).…”

Section: Resultsmentioning

confidence: 99%

Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system

et al. 1993

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A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages.Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNAAGG in the cell to become sequestered in translating peptidyl_tRNAAGG_mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome to frameshift, hop, or terminate translation.Most amino acids are encoded by more than one codon, and frequencies of use of synonymous codons tend to reflect the relative abundance of the tRNAs that recognize them (7,29). Synonymous codons may be translated at different rates (16,17) MATERUILS AND METHODSCodon test plasmids. Plasmids pCT1, pCT2, and pCT3 ( Fig. 1) were assembled from elements of pET vectors (13,24) and of T7 DNA (6). Elements were assembled by using a natural XbaI site (T'CTAGA, where the primer indicates the position of cleavage) between the 410 promoter and the slO translation initiation region for the gene 10 major capsid protein of T7, a natural NdeI site (CA'TATG) at the slO initiation codon, and newly introduced XbaI or NheI (G'CTAGC) sites at the ends of elements. Plasmids were assembled in modular fashion by fusions of compatible ends of DNA fragments from plasmids carrying individual elements or combinations of elements. Most of the fusions were between fragments that had one end at the PstI site in the on May 7, 2018 by guest

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Section: Resultsmentioning

confidence: 99%

Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system

et al. 1993

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“…Processing has been implicated in developmental regulation of rbcL transcript levels in maize and barley, because the amount of -300 RNA relative to -63 RNA increases transiently during greening (49,50). In E. coli, processed mRNAs can be translated with different efficiencies and possess distinct turnover rates relative to their corresponding primary transcript (6,47). Similar mechanisms for the control of rbcL gene expression may operate in chloroplasts of higher plants.…”

Section: Discussionmentioning

confidence: 99%

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

Hanley‐Bowdoin¹,

Orozco²,

Chua³

1985

Mol. Cell. Biol.

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The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.Ribulose 1,5-bisphosphate carboxylase (RUBISCO) catalyzes CO2 fixation, the first step of the Calvin cycle (40). In higher plants, the chloroplast enzyme is composed of eight identical, catalytic polypeptides of 50,000 to 60,000 kilodaltons and eight smaller polypeptides of 12,000 to 20,000 kilodaltons (2). The small subunit, whose function is unknown, is encoded in the nucleus by a small multigene family (7,14,19). The large subunit gene (rbcL) is present as a single copy on the multicopy chloroplast genome (5). Under most conditions, the synthesis of the two RUBISCO subunits is coordinated (55) and regulated by light (15, 50) and cytokinins (23). In maize and other C4 plants, RUBISCO expression is leaf cell-type specific (30). The maize mRNAs encoding the two subunits are present in bundle sheath cells but not in mesophyll cells, which assimilate CO2 via ribulose 1,5-bisphosphate and C4 dicarboxylic acids, respectively (11,35).In chloroplast genomes of higher plants, the genes for the large subunit of RUBISCO and the beta subunit of ATP synthetase (atpB) are adjacent and transcribed divergently (31, 60). The 5' ends of the two genes are separated by approximately 150 base pairs (bp) (52). Northern hybridization analyses of transcripts from a broad spectrum of angiosperms indicate that the rbcL gene is generally transcribed as a 1.6-kilobase mRNA, although larger transcripts are also observed (46). Si nuclease protection experiments have revealed the presence of two 5' termini for the rbcL RNAs of maize, barley, spinach, and peas (16,21,41,49,62.) Tobacco has a single rbcL RNA with its 5' end at position -180 relative to the coding region (53). In maize ...

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“…It is known that stem-loop structures at the 3' termini of bacterial mRNAs can confer enhanced stability to the mRNAs which contain them (9,21,23) or can block what is presumed to be 3' exonucleolytic degradation of trp mRNA terminated at a rho-dependent site (18). By analogy, it seems likely that the rapid degradation of S20-galactokinase fusion mRNA in strain GM291 (terminator negative) ( Table 2) is a consequence of the deletion of the normal S20 transcriptional terminator in pGM46 rather than of translational repression.…”

Section: Methodsmentioning

confidence: 99%

“…The construction used in that experiment deleted a putative rho-independent terminator normally located immediately distal to the gene for S20 (14). The secondary structure found at the 3' end of transcripts generated at rho-independent sites can contribute to the stability of mRNAs so terminated (9,18,21,23). In this work, therefore, I investigated the stability of the native S20 mRNA as a function of growth rate and gene number, factors which should influence translational efficiency.…”

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confidence: 99%

Posttranscriptional regulation of ribosomal protein S20 and stability of the S20 mRNA species

Mackie¹

1987

J Bacteriol

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I have tested whether selective degradation of mRNA for ribosomal protein S20 of Escherichia coli occurs under conditions for which the expression of S20 is regulated posttranscriptionally. Blot hybridization of total RNA extracted from cultures at different times after addition of rifampin has permitted the estimation of relative levels of the two S20 mRNA species and their half-lives. In a strain harboring a plasmid containing the complete gene for S20, including the transcriptional terminator, moderate posttranscriptional repression of S20 synthesis is accompanied by a substantial increase in the half-lives of both S20 mRNAs relative to those in the haploid parental strain. In an otherwise identical strain in which the transcriptional terminator is deleted from the plasmid-borne S20 genes, the half-life of total S20 mRNA declines more than twofold relative to that in the haploid parent. Thus accelerated decay of the mRNAs for ribosomal protein S20 appears to be an artifact of deletion of the transcriptional terminator, rather than a physiologically significant consequence of translational repression.Posttranscriptional regulation appears to be a major mechanism of control of the synthesis of ribosomal protein in Escherichia coli (7,12,19). When the capacity of a cell to synthesize one or more ribosomal proteins exceeds the availability of the nascent rRNA to sequester them, then the translational efficiency of the mRNA(s) encoding the proteins in question decreases. This decline is postulated to be a consequence of the binding of one or more free ribosomal proteins to a site on the mRNA(s) in a manner which reduces the efficiency of translational initiation or, in one instance, leads to premature termination of transcription (28). For ribosomal protein S20, gene dosage experiments (6, 22), the ability of S20 to inhibit its own synthesis in vitro (26,27), and homologies between the leader region of S20 mRNA (deduced from the DNA sequence of its gene) and the 5' third of 16S rRNA (13) are consistent with the basic model for autogenous translational control outlined above.The stability of the mRNAs encoding ribosomal proteins, particularly under conditions of autogenous repression, is a potentially important factor in this control process. Indeed, selective inactivation of the excess mRNA in multicopy strains has been proposed to explain the compensation observed in some ribosomal protein operons for increased gene dosage (4,24). Olsson and Gausing have pointed out, however, that an apparently accelerated rate of decay of an mRNA could be a consequence of its repression rather than the root cause (20). We previously observed that the apparent half-life-of S20 mRNA in a strain carrying 58 copies of the gene decreased to 60 s from 150 s in the haploid parental strain (22). The construction used in that experiment deleted a putative rho-independent terminator normally located immediately distal to the gene for S20 (14). The secondary structure found at the 3' end of transcripts generated at rho-independent sites ca...

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Cleavage within an RNase III site can control mRNA stability and protein synthesisin vivo

Cited by 68 publications

References 21 publications

Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system

Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

Posttranscriptional regulation of ribosomal protein S20 and stability of the S20 mRNA species

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