Coat Polypeptide Research Articles

Effect of Temperature-Sensitive Mutation on Activity of the RNA Transcriptase of Vesicular Stomatitis Virus New Jersey

The virion-associated RNA transcriptase activity of vesicular stomatitis virus New Jersey temperature-sensitive (ts) mutants was assayed in vitro at the permissive (31 degrees C) and restrictive (39 degrees C) temperatures. RNA synthesis at 39 degrees C by the RNA-negative ts A1 and the RNA-positive ts C1 and ts D1 mutants was similar to that of wild-type virus. The RNA-negative ts B1 synthesized only small amounts of RNA in vitro at 39 degrees C. The three mutants of complementation group E were dissimilar in the amounts of RNA they synthesized at 39 degrees C: ts E1 synthesized very little RNA, ts E2 synthesized moderate amounts, and RNA synthesis by ts E3 was not inhibited. The two mutants of group F were also dissimilar, since ts F1 synthesized very little RNA at 39 degrees C, whereas ts F2 synthesized as much RNA as wild-type virus. The revertant clones ts B1/R1, ts E1/R1, and ts F1/R1 synthesized RNA at 39 degrees C in amounts comparable to wild-type virus, indicating that the heat sensitivity of the transcriptase activity of the mutants ts B1, ts E1, and ts F1 was associated with temperature sensitivity. Similar heat sensitivities were observed when transcribing nucleoprotein complexes were used in the assays, showing that the mutated polypeptides were part of the viral core. The heat stability of the mutant ts B1 was similar to that of wild-type virus, and in vitro RNA synthesis was fully restored when the temperature was lowered to 31 degrees C after 30 min of preincubation at 39 degrees C, showing that the inhibition was due to reversible configurational change of the mutated polypeptide. When virions of the mutant ts E1 were heated for 5 h at 39 degrees C, their infectivity and transcriptase activity were as stable as those of the wild-type virus, whereas transcriptase activity became very heat labile after disruption of the viral coat with a neutral detergent. This suggests an interaction between the mutated polypeptide and a coat polypeptide which stabilizes the activity of the transcriptase. The RNA transcriptase activity of the mutant ts F1 was also heat labile, although to a lesser extent than that of ts E1. Thus, the defects in transcriptase activity of groups B, E, and F suggest that all three polypeptides of the virus core, polypeptides L, N, and NS, are involved in the transcription. In addition, we postulate that the mutated gene products of groups E and F are multifunctional, being required both in transcription and replication, and that the gene product of group E may also be involved in some late stage of virus development.

Properties of the Bacillus subtilis spore coat.

About 70% of the protein in isolated Bacillus subtilis spore coats was solubilized by treatment with a combination of reducing and denaturing agents at alkaline pH. The residue, consisting primarily of protein, was insoluble in a variety of reagents. The soluble proteins were resolved into at least seven bands by sodium dodecyl sulfate gel electrophoresis. About one-half of the total was four proteins of 8,000 to 12,000 daltons. These were relatively tyrosine rich, and one was a glycoprotein. There was also a cluster of proteins of about 40,000 daltons and two or three in the 20,000- to 25,000-dalton range. The insoluble fraction had an amino acid composition and N-terminal pattern of amino acids very similar to those of the soluble coat proteins. A major difference was the presence of considerable dityrosine in performic acid-oxidized preparations of insoluble coats. Coat antigen including a 60,000-dalton protein not present in extracts of mature spores was detected in extracts of sporulating cells by immunoprecipitation. This large antigen turned over in a pulse-chase experiment. Antibodies to either the array of 8,000- to 12,000-dalton coat polypeptides or to the larger coat proteins reacted with this 60,000-dalton species, suggesting a common precursor for many of the mature coat polypeptides. Spore coats seem to be assembled by processing of proteins and by secondary modifications including perhaps dityrosine formation for cross-linking.

Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component.

Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer at pH 7 in the presence of protease inhibitors. Approximately 31% of the total spore protein was soluble, and another 14% was removed from the insoluble fraction by hydrolysis with lysozyme and washing with 1 M KCl and 0.1% sodium dodecyl sulfate. The residual spore integuments comprised 55% of the total spore proteins and consisted of coats and residual membrane components. Treatment of integuments with sodium dodecyl sulfate and reducing agents at pH 10 solubilized 40% of the total spore protein. Seven low-molecular-weight polypeptide components of this solubilized fraction comprised 27% of the total spore protein. They are not normal membrane components and reassociated to form fibrillar structures resembling spore coat fragments. The residual insoluble material (15% of the total spore protein) was rich in cysteine and was probably also derived from the spore coats. A solubilized coat polypeptide of molecular weight 12,200 has been purified in good yield (4 to 5% of the total spore protein). Five amino acids account for 92% of its total amino acid residues: glycine, 19%; tyrosine, 31%; proline, 23%; arginine, 13%; and phenylalanine, 6%.

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

Properties of Bacillus cereus temperature-sensitive mutants altered in spore coat formation.

Three conditional Bacillus cereus mutants altered in the assembly or formation of spore coat layers were analyzed. They all grew as well as the wild type in an enriched or minimal medium but produced lysozyme and octanol-sensitive spores at the nonpermissive temperature (35 to 38 degrees C). The spores also germinated slowly when produced at 35 degrees C. Temperature-shift experiments indicated that the defective protein or regulatory signal is expressed at the time of formation of the outer spore coat layers. Revertants regained all wild-type spore properties at frequencies consistent with initial point mutations. Spore coat defects were evident in thin sections and freeze-etch micrographs of mutant spores produced at 35 degrees C. In addition, one mutant contained an extra surface deposit, perhaps unprocessed spore coat precursor protein. A prevalent band of about 65,000 daltons (the same size as the presumptive precursor) was present in spore coat extracts of this mutant and may be incorrectly processed to mature spore coat polypeptides. Another class of mutants was defective in the late uptake of half-cystine residues into spore coats. Such a defect could lead to improper formation of the outer spore coat layers.

Properties of Bacillus cereus spore coat mutants.

Two classes of spore mutants have been selected in Bacillus cereus T, those producing lysozyme-sensitive spores, and those producing spores dependent upon lysozyme for germination. One mutant from each class was studied in detail and found to have defective packing of the spore coat layers. The major spore coat poplypeptide appeared to be altered on the basis of gel electrophoretic profiles and/or peptide maps of half-syctine-containing peptides. The spores of the mutants of both classes were sensitive to lysozyme and failed to respond to the germinants L-alanine plus adenosine. The spores were also more sensitive to octanol than the parental strain, but contained the same amount of dipicolinic acid and were equally heat resistant. The reversion frequencies in both cases were consistent with an initial point mutation, suggesting that an alteration in the major coat polypeptide accounted for the phenotypic properties studied.

Translation of mengovirus RNA in ehrlich ascites cell extracts

Mengovirus RNA was translated in Ehrlich ascites cell extracts using as radioactive precursors f[ 35S]Met tRNA F Met and [ 35S]Met tRNA M Met to label the products in N-terminal and internal positions, respectively. Tryptic peptides were compared with those derived from purified [ 35S]Met-labeled mengovirus. The results indicate that the sequences corresponding to the viral coat polypeptides are preceded by a short “lead-in” peptide which is probably removed by a cleavage process in infected cells.

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.

Recent progress in the sequence determination of bacteriophage MS2 RNA

Summary By hydrolysis with specific ribonucleases we have previously established the terminal sequences of MS-2 RNA as pppG—G—G—U........G—U—U—A—C—C—A—C—C—C—A 3′oh . All sequences of the general structure (Purine nucleotide) n Pyrimidine nucleotide, released by pancreatic ribonuclease hydrolysis and with n ≥ 6 (23 in total) were also sequenced. The longer ones could be assigned either to the right one third of the left two thirds of the entire chain. More recent studies were carried out by partial enzymatic hydrolysis, which allowed the isolation of fragments with chain length 30 to 250. Among these, a 5′-terminal fragment containing 125 nucleotides was identified and completely sequenced. It follows from these results that initiation for protein synthesis at the first cistron, which codes for the A-protein, starts at position 130. The preceding stretch remains untranslated and functions perhaps as a recognition mechanism in replication. The 3′-terminal was isolated as another fragment, which was sequenced up to position ω-70. It does not seem to contain the termination signal for the RNA-polymerase cistron, and suggests that also here an extended, untranslated sequence exists. Five hairpin-like structures, derived from the coat protein cistron, have been isolated and characterized. They can be unambiguously identified, as the sequence of the 129 amino acids of the coat polypeptide is known. These hairpins were subsequently found in still larger fragments. At present 75 p. cent of the nucleotide sequence of this cistron has been established, and only two gaps remain unsolved. All these sequences have a high degree of secondary structure. In the case of the coat cistron, it is clear that in many cases third letters of degenerate codons are chosen on the basis of their ability to participate in base pairing. As expected, the genetic code dictionary, which can now be directly derived from a natural messenger, is in full agreement with the generally accepted code, largely derived from in vitro studies. 35 of the 61 no-nonsense code words have so far been assigned. It is of interest that some degenerate code words seem to be avoided. Another fragment, presumably derived from the polymerase cistron, has also been sequenced. It consists of two polynucleotides, 84 and 34 nucleotides in length respectively. It is of interest, because the outline of the secondary structure seems to be more complex than a simple hairpin. Six more codons, so far not found in the coat cistron, can now be assigned. Some of these, like ACA for threonine, CAA for glutamine and AGU for serine may perhaps play a role in a modulation type control mechanism. Other codons, like AUA for isoleucine and UAU for tyrosine are perhaps not used in Escherichia coli at all.

Replication of Phage RNA

This chapter discusses the replication of the phage of ribonucleic acid (RNA). This chapter discusses the replication of the RNA of the RNA phages. However, reference is made to the replication of other RNA viruses, either to point out similarities or to stress differences. The control mechanisms that regulate the relative amounts of viral proteins and viral RNA species formed are not completely understood. It has been suggested that under normal circumstances, only the parental RNA strands (and possibly early progeny RNA strands) direct the formation of the viral polymerase responsible for the formation of “minus” strands and that the coat polypeptide is involved in translation control. Phage mutants, in which the relevant control mechanisms have been lost, provide a powerful tool for the study of this problem. The replication of most animal and plant viruses, containing single stranded RNA, has many features in common with that of the RNA phages. In particular, it is possible that the replication of viruses, such as Rous sarcoma virus, involves, hitherto, unknown features. Careful analysis of the further purified “RNA replicase” system, which forms infective progeny RNA when primed with parental RNA, will undoubtedly yield a clear answer to this question, as well as to the problem whether one or two separable enzymes are involved in the viral RNA synthesis.

Induction of RNA Synthetase in E. coli after Infection by the RNA Phage, MS2

The discovery of the bacteriophage f2 by Loeb and Zinder (1961), followed by the isolation of similar phages such as MS2 (Davis et al., 1961), FH4 and FH5 (Fouace and Huppert, 1962), Ft5 (Knolle and Kaudewitz, 1962), and r17 (Paranchych and Graham, 1962), offered a new approach to the elucidation of the biology and biochemistry of RNA containing viruses. It was soon established that the protein-free bacteriophage RNA was capable, albeit with low efficiency (10-5 to 10-9), of infecting E. coli protoplasts and causing the formation of normal viral progeny (Knolle and Kaudewitz, 1962; Paranchych, 1963). The exciting finding by Nathans et al. (1962) that f2 phage RNA added to an in vitro protein synthesizing system derived from normal, healthy E. coli would lead to the formation of phage specific coat polypeptides proved that this RNA was not only the repository of viral genetic information but that it could serve...