MS2 Coat Protein Research Articles

Sequence analysis of fluorescamine-stained peptides and proteins purified on a nanomole scale. Application to proteins of bacteriophage MS2.

Some techniques, facilitating the determination of the amino‐acid sequence of proteins or protein fragments available in 10 nanomole amounts are presented.Fluorescamine was used to follow the purification of proteins and peptides without interfering in the determination of the amino‐acid composition and sequence.These techniques were applied on the coat protein of coliphage MS2 and on some peptides obtained from a chymotryptic digest of the assembly protein of MS2.

A combinatorial study of the effects of admitting non-watson-crick base pairings and of base composition on the helix-forming potential of polynucleotides of random sequence

The development of extensive double-helical secondary structure in random polynucleotides depends on the existence of regions of complementary or nearly complementary base sequence. Although the precise structure assumed in solution by a particular polynucleotide is determined by thermodynamic considerations, a purely statistical investigation of the expected extent of complementarity can yield useful general insights. Such probabilistic techniques are applied to a study of the effects of base composition and of different assumed base-pairing schemes on the extent of secondary structural development in random polynucleotides. The results suggest limitations on the possible evolutionary precursors to the extensive and nearly perfect helical structure exhibited by nucleic acids, such as the cloverleaf of tRNA and the flower model of the MS2 coat protein gene.

IF3 - interference factors : protein factors in Escherichia coli controlling initiation of mRNA translation

Summary Initiation factor IF3 activity can be separated into subfractions differing by their relative activity for various mRNA templates. In most cases, it can be shown that the template specificity of these subfractions results from the presence of other protein factors, having no IF3 activity by themselves, but which interfere with IF3 activity. Purified IF3 is active for both MS2 and T4 mRNA, but on MS2 RNA it stimulates selectively initiation of the coat protein cistron. Addition of interference factor i to this standard IF3, blocks initiation at the MS2 coat protein cistron, but stimulates it at the MS2 synhetase cistron. Moreover, this protein of MW 74,0000, becomes after RNA phage infection a subunit of the RNA replicase. Factor i stimulates also T7 mRNA translation. On T4 mRNA, it stimulates the translation of certain cistrons, while inhibiting that of others. In addition to factor i, other interference factors have been detected. Factor J, for example, inhibits MS2 coat and synthetase cistrons ; it inhibits T7 mRNA translation and produces a higher inhibition of early T4 mRNA than late T4 mRNA. Another interference factor inhibits late T4 mRNA more than MS2 RNA. In combination with IF3, these proteins give a series of initiation factors with very different template specificities. They bind to ribosomes and IF3 and confer to the ribosome initiation complex a selective affinity for certain initiation signals. After T4 infection, the changes in ribosome template specificity may result from a change either in IF3 or in an interference factor. Immunological and biochemical data indicate that IF3 is unchanged. However interference factor i is chemically modified after T4 infection. Infection of E. coli F+ strains by phage T7 results in a rapid loss of IF3 from the cell, which may be responsible for the absence of late protein synthesis and for the abortive T7 infection in these strains. This does not happen in E. coli F− strains. In stationary phase E. coli, there is a partial loss of IF3 : this decreases the ratio of IF3 to interference factor i and alters the relative translation of T4 and MS2 RNA. From these examples, it is concluded that IF3-interference factors participate in the control of mRNA translation.

Amino-acid sequence invariance and genetic code.

FROM the complete nucleotide sequence of the gene coding for the coat protein of coliphage MS2, Min Jou et al.1 have suggested a scheme for base-pairing between certain stretches of the polynucleotide chain. Their hypothesis leads to the formation of double helices in their “flower” model and to the consequent implication that nucleic acids have a well-defined tertiary structure. Such a scheme remains to be proved by direct observation of the structure, but the indirect arguments of Min Jou et al. and others appear so convincing and the notion that the genetic material has a definite tertiary structure seems so probable, that there can be little doubt that the “flower” model is on the right lines in principle, even though it may be incorrect in detail.

Cistron specific translation control protein in Escherichia coli.

GENE expression may be controlled during translation by ribosomal selection of mRNAs or even individual cistrons. Escherichia coli initiation factors associated with ribosomes affect the binding of ribosomes to mRNA1,2; initiation factor IF3, for instance, influences the specificity of mRNA-ribosome interaction3,4. IF3 activity has been separated into several fractions which show various specificities for different mRNA cistrons4–9. An important problem is the possibility of intracellular changes in IF3 activity10–12. From uninfected E. coli, we have now isolated a protein which changes the specificity of IF3 toward different mRNAs; we call this interference factor i. Pure factor i binds to IF3 and specifically affects the translation of T4 and MS2 RNA. Whereas the initiation of translation of the MS2 coat protein cistron is inhibited by factor i, the synthetase cistron—when available—is more rapidly initiated in the presence of factor i. The overall translation of T4 mRNA appears unchanged by factor i, but certain cistrons are stimulated at the expense of others. Interfering factors such as factor i could be important in controlling translation in E. coli.

Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein.

By characterization of fragments, isolated from a nuclease digest of MS2 RNA, the entire nucleotide sequence of the coat gene was established. A “flower”-like model is proposed for the secondary structure. The genetic code makes use of 49 different codons to specify the sequence of the 129 amino-acids long coat polypeptide.

Translational control of bacteriophage MS2 RNA cistrons by MS2 coat protein: Affinity and specificity of the interaction of MS2 coat protein with MS2 RNA

The effect of MS2 coat protein on MS2-specific protein synthesis was studied in vitro. Protein synthesis was initiated by adding MS2 RNA to the in vitro protein-synthesizing system and MS2 coat protein was added to the system at varying time intervals afterwards. The addition of coat protein specifically reduced the rate of viral RNA polymerase synthesis (assayed by histidine incorporation) without significantly affecting the rate of coat protein synthesis (assayed by phenylalanine incorporation). The addition of coat protein to the on-going system rapidly reduced histidine incorporation to a rate comparable to that which occurred when coat protein was incubated with MS2 RNA (to form “complex I”) prior to their addition to the protein-synthesizing system. The concentrations of MS2 RNA and MS2 coat protein used in one experiment were quite low and probably in the range of their actual concentrations in the cytoplasm of the infected host cells. These results indicate (1) that the interaction of MS2 coat protein and MS2 RNA is highly specific, since various cellular components present in the protein-synthesizing system did not interfere with the effect of the added coat protein and (2) that the affinity of coat protein for MS2 RNA is high enough for the formation of complex I to take place at the relatively low concentrations of MS2 RNA and coat protein presumed to occur in the cytoplasm of the infected cells.

Regulation of protein synthesis directed by coliphage MS2 RNA: II. In vitro repression by phage coat protein

In vitro protein synthesis directed by bacteriophage MS2 RNA is inhibited by prior incubation of the RNA with MS2 coat protein. This inhibition involves exclusively non-coat proteins as shown by the selective decrease in incorporation of histidine, which is absent in phage coat protein, and by the results of acrylamide gel fractionation of the cell-free protein products. The specificity of the inhibitory effect is shown by the failure of MS2 coat protein to inhibit protein synthesis directed by coliphage Qβ RNA, by the lack of activity of Qβ coat protein with MS2 RNA, and by the indiscriminate inhibition of phage protein synthesis by the polycation polylysine. Coat protein inhibition was correlated with the formation of a complex between MS2 RNA and a small number of MS2 coat protein molecules (complex I of Sugiyama, Hebert & Hartman, 1967). Qβ RNA failed to form such a complex with MS2 coat protein, although it did form a complex with a high protein to RNA ratio. On the basis of these observations we suggest that coat protein inhibition results from the binding of a small number of protein molecules to the phage RNA at one or more sites outside the coat protein gene and prevents the translation of the other phage genes.

Regulation of protein synthesis directed by coliphage MS2 RNA: I. Phage protein and RNA synthesis in cells infected with suppressible mutants

In actinomycin-treated Escherichia coli infected with coliphage MS2 three phage-specific proteins have been detected after acrylamide gel electrophoresia. These proteins have been identified by the use of Suppressible mutants of MS2 as the phage coat protein, “maturation” or assembly protein and RNA synthetase. In MS2-infected cells RNA synthetase appeared before the other two proteins were detectable. All three proteins then increased, but the synthesis of maturation protein and RNA synthetase rapidly declined between 30 and 40 minutes post-infection, whereas the synthesis of coat protein continued at a high rate throughout the infectious cycle. The synthesis of phage RNA paralleled that of the RNA synthetase and maturation protein. Non-permissive cells infected with suppressible RNA synthetase mutants made no detectable phage proteins. Cells infected with maturation protein mutants showed a normal pattern of coat protein and RNA synthetase formation. Infection with a coat protein mutant resulted in a greater than normal rate of formation of RNA synthetase and maturation protein relative to the amount of phage RNA, and a higher percentage of each of these proteins was made late in infection compared to the situation in MS2-infected cells. However, the increase of these proteins was not co-ordinate: RNA synthetase formation increased more than the synthesis of maturation protein. In cells doubly infected with a maturation protein mutant and a coat protein mutant the derepression of synthetase and maturation protein seen in cells singly infected with the coat mutant was largely reversed. Our major conclusion is that the coat protein of MS2 serves as a repressor of the synthesis of maturation protein and RNA synthetase.

Translational control of bacteriophage MS2 RNA cistrons by MS2 coat protein: Polyacrylamide gel electrophoretic analysis of proteins synthesized in vitro

MS2-specific proteins synthesized in a cell-free, protein-synthesizing system under the direction of MS2 RNA were analyzed by polyacrylamide gel electrophoresis in a buffer containing sodium dodecyl sulfate. In addition to coat protein, which accounted for half or more of the total [ 14C]phenylalanine-labeled proteins synthesized, a few non-coat proteins were resolved. When a ribonucleoprotein complex (termed complex I), formed in vitro by interaction of MS2 RNA with several molecules of MS2 coat protein, was used instead of MS2 RNA to direct in vitro protein synthesis, the synthesis of some non-coat proteins was greatly reduced, whereas the synthesis of coat protein was almost unaffected. These results support our previous proposition that complex I is involved in the mechanism for the shut-off of synthesis of non-coat proteins in MS2-infected cells. In the cell-free, protein-synthesizing system, coat protein appeared to be the first protein synthesized. This is consistent with the notion that the coat protein cistron in MS2 RNA is located close to the initiation point of translation.

Demonstration of further differences between in vitro and in vivo synthesized MS2 coat protein.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDemonstration of Further Differences between in Vitro and in Vivo Synthesized MS2 Coat Protein*Jung-Yaw Lin and H. Fraenkel-ConratCite this: Biochemistry 1967, 6, 11, 3402–3407Publication Date (Print):November 1, 1967Publication History Published online1 May 2002Published inissue 1 November 1967https://doi.org/10.1021/bi00863a010RIGHTS & PERMISSIONSArticle Views20Altmetric-Citations4LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (468 KB) Get e-Alerts Get e-Alerts

Control of translation of MS2 RNA cistrons by MS2 coat protein.

Control of translation of MS2 RNA cistrons by MS2 coat protein.

The coat protein of the RNA bacteriophage MS2

The amino acid composition of the coat protein of the MS2 phage seems to be identical to that of M12. The f2 phage differs from these by one methionine to leucine exchange and the R17, seemingly, by a glutamic acid or amide to lysine exchange. Alanine was found to be the N-terminal amino acid, and tyrosine preceded by isoleucine the C-terminal amino acids of the MS2 protein. All 11 unique tryptic fragments of the MS-2 protein have been separated and analyzed. By amino ethylation, followed by tryptic digestion, it was possible to locate the two cysteine residues and by cyanogen bromide cleavage the two methionine residues. The latter technique has been of particular advantage in lessening the interaction of the core peptides obtained upon tryptic digestion, and facilitating the isolation of the biggest peptide. Cyanogen bromide cleavage, in conjunction with the isolation of peptides resulting from subsequent thermolysin digestion, has also made it possible to align all tryptic peptides in a unique sequence.