A-Protein gene of bacteriophage MS2

Fiers, Walter; Contreras, Roland; Duerinck, F; Haegmean, G; Merregaert, J.; Jou, Willy Min; Raeymakers, Alex; Volckaert, Guido; Ysebaert, Marc; Kerckhove, Joris Van; Nolf, F; Montagu, Marc Van

doi:10.1038/256273a0

Cited by 156 publications

(27 citation statements)

References 43 publications

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“…3). The wild-type repressor protein is initiated with GUG at nucleotide 29. This demonstrates that, at a normal initiation site in an E. coli mRNA, GUG can specify N-formylmethionine; previously, GUG was identified as the initiator triplet for the bacteriophage MS2 A protein (17). The I mRNA sequence also confirms the earlier assignment of GUG and AUG to amino acid positions 23 and 42, where they function as initiators for the first two restart polypeptides.…”

Section: Discussionsupporting

confidence: 60%

5′-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: Features of translational initiation and reinitiation sites

Steege

1977

Proc. Natl. Acad. Sci. U.S.A.

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In a sequence of 214 nucleotides at the 5' terminus of the Igene mRNA, which codes for the lactose repressor protein of Escherichia coli, (i) an untranslated leader sequence of 28 residues precedes the repressor coding region; (ii) a GUG initiates synthesis of the wild-type repressor; (iii) GUG and AUG are the functional initiators for the synthesis of restart polypeptides activated by early I gene amber mutations, confirming previous assignments for these residues based on protein sequencing data; and (iv) sequences complementary to 16S ribosomal RNA provide stronger potential mRNA-16S rRNA interaction at the wild-type initiation site than at the restart sites. When I mRNA is used to direct the formation of initiation complexes in vitro, ribosomes bind only to the wild-type initiator region.A striking feature of the I mRNA sequence is the presence of a number of in-phase GUGs that have not been observed to serve as initiation signals in vivo in the nonsense mutant strains examined. The selective use of potential initiator triplets in the I mRNA leads to the following conclusions. First, when presented with several neighboring initiator triplets at the wild-type initiator region, ribosomes select the one preceded by the strongest appropriately positioned complementarity to the 16S 31 end. Second, ribosomes do not restart after termination simply by moving to the next available initiator codon. Third, the formation of stable secondary structures predicted for the untranslated I mRNA beyond chain-terminating nonsense mutations may prevent ribosome access to some potential reinitiation sites.The lactose repressor protein, encoded by the lac I gene, exerts negative control over the expression of the lactose operon of Escherichia coli. The possibility of using the I gene to obtain information concerning the specificity of translation initiation was raised several years ago by the discovery that nonsense mutations early in the gene activate reinitiation of repressor synthesis beyond the position of the chain terminating codon. At least three sites within that portion of the mRNA corresponding to the first 62 amino acids of the wild-type repressor protein direct the synthesis of COOH-terminal restart polypeptides which accumulate at 10% of the wild-type repressor level (1-3).The amino acid sequences of the wild-type repressor (4, 5) and the restart polypeptides from amber mutant strains (1-3) predict which I mRNA codons are utilized as reinitiation signals. Whereas restart proteins are not detected in wild-type cells, two restart polypeptides are found in strains carrying amber mutations at amino acid positions 7, 12, and 17. The larger one initiates at a valine codon (presumed to be GUG) in position 23; the smaller one initiates at a methionine codon (AUG) in position 42. When the amber block is moved beyond the first restart

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Section: Discussionsupporting

confidence: 60%

5′-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: Features of translational initiation and reinitiation sites

Steege

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…Both the degree of secondary structure and the level of genomic association with proteins are known to influence genomic susceptibility to degradation. The genome of MS2 has a complex secondary structure (12,13) and is connected to the capsid at both the 5Ј and 3Ј ends via the A protein (36). Both of these factors have been shown to confer a higher resistance to ribonucleases and may explain why certain regions are more or less susceptible than others.…”

Section: Susceptibility Of Different Genome Regions To Degradationmentioning

confidence: 99%

Quantitative PCR for Determining the Infectivity of Bacteriophage MS2 upon Inactivation by Heat, UV-B Radiation, and Singlet Oxygen: Advantages and Limitations of an Enzymatic Treatment To Reduce False-Positive Results

Pecson

Martin

Kohn

2009

Appl Environ Microbiol

161

168

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Health risks posed by waterborne viruses are difficult to assess because it is tedious or impossible to determine the infectivity of many viruses. Recent studies hypothesized that quantitative PCR (qPCR) could selectively quantify infective viruses if preceded by an enzymatic treatment (ET) to reduce confounding false-positive signals. The goal of this study was to determine if ET with qPCR (ET-qPCR) can be used to accurately quantify the infectivity of the human viral surrogate bacteriophage MS2 upon partial inactivation by three treatments (heating at 72°C, singlet oxygen, and UV radiation). Viruses were inactivated in buffered solutions and a lake water sample and assayed with culturing, qPCR, and ET-qPCR. To ensure that inactivating genome damage was fully captured, primer sets that covered the entire coding region were used. The susceptibility of different genome regions and the maximum genomic damage after each inactivating treatment were compared. We found that (i) qPCR alone caused false-positive results for all treatments, (ii) ET-qPCR significantly reduced (up to >5.2 log units) but did not eliminate the false-positive signals, and (iii) the elimination of false-positive signals differed between inactivating treatments. By assaying the whole coding region, we demonstrated that genome damage only partially accounts for virus inactivation. The possibility of achieving complete accordance between culture-and PCR-based assays is therefore called into doubt. Despite these differences, we postulate that ET-qPCR can track infectivity, given that decreases in infectivity were always accompanied by dose-dependent decreases in ET-qPCR signal. By decreasing false-positive signals, ET-qPCR improved the detection of infectivity loss relative to qPCR.Water-and food-borne viruses are a major worldwide source of gastroenteritis, and thus the detection and inactivation of infective viruses are important public health priorities. Cell culture can be employed to quantify the infectivity of certain viruses. However, culturing can take days to weeks to yield results, and many viruses important to public health, e.g., hepatitis A and noroviruses, are either difficult to culture or are nonculturable (3,19,38). Immunological and spectrometryand microscopy-based methods have also been developed, but none of them has provided a satisfactory alternative (5,26,43,45).The advent of PCR and quantitative PCR (qPCR) in environmental microbiology had raised hopes that these limitations would be overcome. While PCR lives up to its promise to provide rapid, sensitive, and specific detection of environmental viruses, its ability to differentiate infective from inactivated viruses has not been realized. This is mainly due to the persistence of genomes of inactivated viruses that remain either partially or fully intact. During PCR, intact genomic regions of inactivated viruses are amplified and produce confounding false-positive PCR signals (3,11,37,38,41). These false positives inhibit our ability to use qPCR to obtain quantitative inform...

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“…Since the presence of unlabeled pools of these amino acids within the cell would alter the ratio from that externally added, we used strains of cells unable to synthesize both leucine and histidine. By purifying doubly labeled A protein from phage and measuring the amount of and 14C associated with it, we were able to calculate the true ratio of the specific activities within newly made protein, since A protein is known to contain 5 histidines for every 36 leucines [38]. Two determinations were made on polyamine-grown cells using method A; the specific activity of added ['4C]leucine was increased to compensate for the fact that less A protein than coat was purified from phage.…”

Section: Specific Activity Of Amino Acids Within Cellmentioning

confidence: 99%

Effect of polyamines on translation fidelity in vivo

McMurry

Algranati

1986

European Journal of Biochemistry

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Polyamines, when added to cell-free protein-synthesizing systems, have been shown either to reduce mistranslation or to increase it depending upon the composition of the reaction mixture. To study this question under conditions as natural as possible we investigated the effects of polyamines on the fidelity of protein synthesis in intact Escherichiu coli bacterial cells, using strains which were auxotrophic for polyamine synthesis. Error was measured in two ways: (a) the incorporation of [3H]histidine into coat protein of bacteriophage MS2, the gene of which does not code for histidine, and (b) the synthesis of a basic variant of MS2 coat protein in which a lysine erroneously replaces an asparagine, causing a change in isoelectric point. We found that when cell cultures were supplemented with polyamines there was no effect on the first type of error and the second type decreased twofold. The measured errors occurred at the level of translation because their frequency increased in the presence of streptomycin and decreased in cells bearing a streptomycin-resistance mutation known to lower the occurrence of translational misreading. The average erroneous incorporation per mol coat protein in the presence of polyamines was 1.43 ? 0.59 mmol histidine and 25 -34 mmol lysine/asparagine substitution. The reason for the different effect of polyamines on the two types of error is not known but could be related to the difference between their corresponding frequencies or to codon-specific effects.Polyamines are widely distributed organic cations which appear to have many roles, especially in processes involving nucleic acids, such as DNA replication, transcription and translation [I -51. Studies carried out with cell-free systems have demonstrated that polyamines can replace part of the magnesium requirement of protein synthesis and at the same time cause overall stimulation [6 -81. Polyamines have also been shown to have a qualitative effect on the proteins synthesized. Adenovirus proteins made in vitro in the presence of polyamines resembled more closely those synthesized in vivo, both in size and quantity [9]. Polypeptides of higher molecular mass were found in smaller amounts in the absence of polyamines in vitro [lo, 111 and in vivo [12]. These observations had suggested that polyamines might prevent premature peptide termination or otherwise affect the fidelity of translation. It was subsequently reported that polyamines, particularly spermidine, decreased the misincorporation of leucine for phenylalanine severalfold in a polyuridylic-acid-dependent in vitro translation system [13-151. However, in these studies the error determinations in presence of polyamines were done at lower Mg concentrations than those in the absence of polyamines because the Mg optimum for polyphenylalanine synthesis was higher under the latter conditions than in the presence of polyamines. In one study, other changes besides polyamine addition were made [16]. At some concentrations of Mg, however, the error was no different or [18] that when...

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A-Protein gene of bacteriophage MS2

Cited by 156 publications

References 43 publications

5′-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: Features of translational initiation and reinitiation sites

5′-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: Features of translational initiation and reinitiation sites

Quantitative PCR for Determining the Infectivity of Bacteriophage MS2 upon Inactivation by Heat, UV-B Radiation, and Singlet Oxygen: Advantages and Limitations of an Enzymatic Treatment To Reduce False-Positive Results

Effect of polyamines on translation fidelity in vivo

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