Coat Protein Cistron Research Articles

Translation of red clover necrotic mosaic virus RNA in rabbit reticulocyte lysate: Identification of the virus coat protein cistron on the larger rna strand of the bipartite genome

The bipartite genome of red clover necrotic mosaic virus (RCNMV) was translated in nuclease-treated rabbit reticulocyte lysate with the synthesis of three principal translation products of molecular weights 39,000, 36,000, and 34,000. The 39,000 translation product was shown to be RCNMV coat protein by peptide mapping after partial proteolysis and by specific immunoprecipitation. RCNMV coat protein was shown to be coded for on the larger RNA (RNA-1) of the bipartite genome by hybrid-arrested translation using complementary DNA prepared to each of the fractionated RNAS. The 36,000-molecular weight product was also translated from RNA-1 while the 34,000-molecular weight product was not. Preliminary evidence indicated that the RCNMV coat protein is translated from a subgenomic species of RNA-1.

The tRNA-like structure of turnip yellow mosaic virus RNA: structural organization of the last 159 nucleotides from the 3′ OH terminus

The secondary structure of the isolated tRNA-like sequence (n=159) present at the 3' OH terminus of turnip yellow mosaic virus RNA has been established from partial nuclease digestion with S1 nuclease and T1, CL(3), and Naja oxiana RNases. The fragment folds into a 6-armed structure with two main domains. The first domain, of loose structure and nearest the 5' OH terminus, is composed of one large arm which extends into the coat protein cistron. The second, more compact domain, is composed of the five other arms and most probably contains the structure recognized by valyl-tRNA synthetase. In this domain three successive arms strikingly resemble the T[unk], anticodon, and D arms found in tRNA. Near the amino-acid accepting terminus, however, there is a new stem and loop region not found in standard tRNA. This secondary structure is compatible with a L-shaped three-dimensional organization in which the corner of the L and the anticodon-containing limb are similar to, and the amino-acid accepting region different from, that in tRNA. Ethylnitrosourea accessibility studies have shown similar tertiary structure features in the T[unk] loop of tRNA and in the homologous region of the viral RNA.

Evidence that RNA 4 of alfalfa mosaic virus does not replicate autonomously

A mutant (Tb ts7) of alfalfa mosaic virus, the coat protein of which is unable to activate the viral genome (the RNA species 1, 2, and 3, which need some coat protein for infectivity) at 30°, can be rescued at this temperature by adding to the inoculum wild-type RNA 3 (the genome part that contains the coat protein cistron), but not adding wild-type RNA 4 (the subgenomic messenger for the coat protein). Unless RNA 3 of Tb ts 7 has a second ts mutation at a site not occurring in RNA 4, it may be concluded from the above finding that RNA 4 does not replicate autonomously.

A comparative study of the cowpea and bean strains of southern bean mosaic virus

Features of the primary structure and translation of the genomic RNAs of the cowpea and bean strains of southern bean mosaic virus have been investigated in order to assess the similarity of the two viruses. The sequence of 400 bases at their 3′ termini have been determined. These include the 3′ noncoding regions and extend well into the coat protein cistrons. The noncoding regions (136 bases for the cowpea strain RNA and 129 bases for the bean strain RNA) show no obvious sequence homology. However, extensive base as well as amino acid sequence homology exists in the coding region. RNAs from both strains have a small protein attached to their 5′ terminus—the protein in the cowpea strain being the smaller of the two. In vitro studies show that there are similarities in the overall mode of translation of the genomes of the two viruses. Although corresponding proteins are synthesized they differ in size.

Nucleotide sequence of a cloned cDNA copy of TMV (cowpea strain) RNA, including the assembly origin, the coat protein cistron, and the 3' non-coding region.

The cloned cDNA derived from the 3' end of cowpea strain (Cc) RNA of tobacco mosaic virus (TMV) has been sequenced. Substantial sequence information of 1,060 nucleotides from the 3' end of the RNA reveals some interesting features: (1) the coat protein cistron corresponds to residues 210-701 from the 3' end. Some errors in the amino acid sequence previously reported have been corrected and the revised total length of the coat protein is 162 amino acid residues. The capping site of the coat protein mRNA is at residue 711 from the 3' end of genome RNA. (2) The assembly origin of reconstitution is positioned within the coat protein cistron at residue 369-461 which can be formed into a highly base-paired hairpin loop structure. The sequence, GAXGUUG, in the loop region and a triplet-repeated purine base tract surrounding the loop are found. These structural features are common to assembly origins of both Cc and vulgare strains. (3) We find the sequence highly homologous to, but distinct from, the genuine assembly origin. It will be called the pseudo-assembly origin, which is located in the corresponding region to the assembly origin of the vulgare strain, outside the coat protein cistron. There is also the sequence, GAXGUUG, in the middle of the region. (4) In the 5' flanking region of the coat protein cistron, a long reading frame, probably of 30 K protein, is found. The coding region is terminated in the coat protein cistron and thus the 30 K protein and the coat protein cistrons overlap. (5) The 3' non-coding region is 209 residues long and can be folded into a possible tRNA-like structure. Surprisingly, we find that the 3' terminal sequence of Cc RNA is not very similar to that of vulgare RNA but extensively homologous to that of turnip yellow mosaic virus (TYMV) RNA.

Complete nucleotide sequence of brome mosaic virus RNA3

A complementary DNA copy of the 3a cistron of dicistronic RNA3 from brome mosaic virus has been cloned and sequenced. Based on this and other data, the complete nucleotide sequence of brome mosaic virus RNA3 is presented. Following a 91 base 5′ non-coding region, the 909 base 3a cistron encodes a protein of predicted molecular weight 32,480. Between the 3a and coat protein cistrons is an approximately 250 base intercistronic non-coding region. Overlapping the intercistronic region by 9 bases, the last 876 bases of RNA3 encode the subgenomic coat protein messenger, RNA4, completely and without interruption. A third long open reading frame of possible evolutionary significance overlaps the 3a and coat protein cistrons. An internal poly(A) of heterogeneous length (16 to 22 bases) occurs in the intercistronic region just 20 bases 5′ to the start of RNA4 sequences. Averaging the poly(A) heterogeneity, the length of brome mosaic virus RNA3 is 2114 bases.

Amino acid sequence of the coat protein subunit in satellite tobacco necrosis virus

The primary structure of the coat protein subunit in satellite tobacco necrosis virus has been investigated. The results obtained are consistent with and support the proposal for the amino acid sequence made from the nucleotide sequence of RNA (Ysebaert et al., 1980). This would imply that no intervening sequences of RNA occur in the cistron for the satellite tobacco necrosis virus coat protein. The polypeptide chain of the protein consists of 195 amino acid residues. It contains one sulfhydryl group but no disulfide bridges. The distribution of various kinds of amino acid residues along the chain is markedly uneven.

Correlation between particle multiplicity and location on virion RNA of the assembly initiation site for viruses of the tobacco mosaic virus group

The initiation site for reconstitution on genome RNA was determined by electron microscopic serology for a watermelon strain of cucumber green mottle mosaic virus (CGMMV-W), which is chemically and serologically related to tobacco mosaic virus (TMV). The initiation site was located at the same position as that of the cowpea strain, a virus that produces short rods of encapsidated subgenomic messenger RNA for the coat protein (a two-component TMV), being about 320 nucleotides away from the 3' terminus, and hence within the coat protein cistron. Although CGMMV-W was until now believed to be a single-component TMV, the location of the initiation site indicated the presence of short rods containing coat protein messenger RNA in CGMMV-W-infected tissue, as in the case for the cowpea strain. We found such short rods in CGMMV-W-infected tissue. The results confirmed our previous hypothesis that the site of the initiation region for reconstitution determines the rod multiplicity of TMV. The finding of the second two-component TMV, CGMMV, indicates that the cowpea strain of TMV is not unique in being a two-component virus and that the location of the assembly initiation site on the genome RNA can be a criterion for grouping of viruses.

An homologous in vitro assay for yeast nonsense suppressors.

A cell-free translation system, from the yeast Saccharomyces cerevisiae, has been used to develop an in vitro assay for yeast UGA, ochre and amber suppressors. Amber suppression was assayed by read-through of the brome mosaic virus coat protein cistron UAG terminator. UGA suppression was assayed by read-through of the rabbit beta-globin UGA terminator and ochre suppression by read-through of the rabbit alpha-globin mRNA UAA terminator. Ochre suppression was increased 3-fold when the globin mRNA was heat denatured prior to translation; this was due to an increase in the synthesis of alpha-globin relative to beta-globin. Amber suppression was more efficient in vitro (46%) than ochre suppression (14%). UGA suppression was also highly efficient in vitro, reaching almost 100% using a purified UGA suppressor tRNA from Schizosaccharomyces pombe. Unfractionated yeast tRNA, from a sup+ strain, contained a tRNA species able to suppress UGA termination codons in vitro, but no tRNA species able to suppress either UAA or UAG was found. This homologous in vitro assay for yeast nonsense suppressors will allow, for the first time, an approach to the biochemical analysis of yeast mutants that modify the efficiency of nonsense suppression in vivo.

Limited sequence variation in the leader sequence of RNA 4 from several strains of alfalfa mosaic virus

The sequences of the 5′-noncoding regions of RNA 4 of seven strains of alfalfa mosaic virus were compared. In each case, the coat protein cistron was preceded by a leader sequence of 39 nucleotides (including the initiator codon). At position 26, an A-, G-, or U-residue was found. Otherwise the leader sequences are identical.

Nucleotide sequence of cauliflower mosaic virus DNA

The complete nucleotide sequence (8024 nucleotides) of the circular double-stranded DNA of cauli-flower mosaic virus has been established. The DNA molecule is known to possess three discrete single-stranded discontinuities, often referred to as “gaps”, two in one strand and one in the other. The sequence data indicate that gap 1, the single discontinuity in the α strand, corresponds to the absence of no more than one or two nucleotides with respect to the complementary β strand. The two discontinuities in the β strand, however, are not authentic gaps since no nucleotides are missing, but are instead regions of sequence overlap: a short sequence (19 residues for gap 2, at least 2 residues for gap 3) at one terminus of each discontinuity, probably the 5′ terminus, is displaced from the double helix by an identical sequence at the other boundary of the discontinuity. Analysis of the distribution of nonsense codons in the DNA sequence is consistent with other evidence that only the α strand is transcribed. The coding region extends around the circular molecule from 4 map units of gap 1, the map origin, to map position 91, and consists of six long open reading frames. Our findings suggest, but do not prove, that the DNA sequence of the open reading frames is colinear with viral protein sequences. The cistron for the viral coat protein, which is probably synthesized in the form of a precursor, has been situated in coding region IV on the basis of its unusual amino acid composition.

Sequence of the 3′ untranslated region of brome mosaic virus coat protein messenger RNA

The 3′ terminal 337 bases of BMV (brome mosaic virus) coat protein mRNA (BMV RNA4) are presented. This sequence includes the terminal portion of the coat protein cistron and the complete 300-base 3′ noncoding sequence. The 3′ noncoding sequence displays significant complementarity to the 5′ terminal sequence of BMV RNA3 but not to the 5′ terminal sequence of BMV RNA4.

Aberrations of the classic codon reading scheme during protein synthesis in vitro.

Using a protein synthesizing in vitro system programmed with MS2-RNA, the ability of alanine tRNAs with the anticodons U*GC (U* represents 5-oxyacetic acid uridine monophosphate) and IGC to read the alanine codons in the coat protein cistron of MS2 has been determined both under conditions of no competition, where the alanyl-tRNA used was the only aminoacylated tRNAAla present in the system, and in experiments where the two alanyl-tRNAs were competing against each other. Under conditions of no competition, each of the anticodons can read all four alanine codons. However, when the anticodons compete for the codon GCC, the anticodon IGC, which can read all three positions of the codon according to the rules of Watson-Crick base pairing, is considerably more efficient than U*GC, which misreads the codon by reading only the first two positions and presumably disregards the third nucleotide of the codon. The outcome of the competition experiments also reveals two apparent violations of the wobble restrictions: the anticodon U*GC reads the codon GUU almost as effectively as does the anticodon IGC, and IGC is almost as effective as U*GC in reading the codon GCG.

The site of initiation of rod assembly on the RNA of a tomato and a cowpea strain of tobacco mosaic virus

Genome RNA of a tomato (T) and a cowpea (Cc) strain of tobacco mosaic virus (TMV), as well as RNA of the short particles of cowpea strain, were tritium labeled at the termini. Chromatographic analyis of the alkali-hydrolysate of these RNAs showed that they all have A OH at the 3′-terminus and are capped with m 7G ppp at the 5′-terminus. The site of initiation of rod assembly for these strains was determined by sequential reconstitution of virus rods with proteins of two different strains followed by examination of distribution of the proteins on the reconstituted rods by electron microscopic serology. The initiation site on T-RNA was located at the same position as that of a common strain previously studied, being about 800 nucleotides away from the 3′-terminus. In contrast, the initiation site on Cc-RNA was found to be much closer to the 3′-terminus, only about 320 nucleotides away from the terminus, and hence within the coat protein cistron. The results showed that the internal initiation and the bidirectional elongation are a universal mechanism of assembly among TMV strains, but different strains may use different initiation sites. The location of the initiation site of the cowpea strain explained why the coat protein messenger RNA of this strain, but not that of the common and the tomato strains, is encapsidated to form short particles.

Translation Products of Genome and Satellite RNAs of Tomato Black Ring Virus

SUMMARY In wheat germ extracts and reticulocyte lysates the genome RNA molecules of tomato black ring virus (TBRV), RNA-1 (mol. wt. 2.8 × 106) and RNA-2 (mol. wt. 1.6 × 106), were translated into products of maximum mol. wt. 2.2 × 105 and 1.6 × 105, respectively. These products represent about 80% and 100% of the coding capacity of the two RNA species. The 1.6 × 105 mol. wt. polypeptide reacted with antiserum to TBRV particles but the translation products of RNA-1 did not; this is additional evidence that RNA-2 contains the coat protein cistron. Satellite RNA molecules (RNA-3, mol. wt. 4.8 × 105) associated with strain S of TBRV, like those associated with strain G, were translated in wheat germ extracts into a polypeptide of mol. wt. 4.8 × 104; this did not react with TBRV antiserum. Protease digestion released peptides from the translation product of strain S RNA-3 which were different from those released from the translation product of strain G RNA-3, suggesting that the two kinds of satellite RNA molecules differ in base sequence.

Initiation site for assembly of several strains of TMV and its relation to occurrence of the short particles in infected plants

The site of initiation of rod assembly for a tomato (T) and a cowpea (Cc) strain of tobacco mosaic virus and for cucumber green mottle mosaic virus (CGMMV) was determined by electron microscopic serology. The initiation site on T-RNA was located at the same position as that of a common strain previously studied, being about 800 nucleotides away from the 3′-terminus. In contrast, the initiation site on Cc-RNA and CGMMV-RNA was found to be much closer to the 3′-terminus, only about 320 nucleotides away from the terminus, and hence within the coat protein cistron. The location of the initiation site of Cc and CGMMV explained why the coat protein messenger RNA of these strains, but not that of the common and the T strains, is encapsidated to form short particles.

Enhancing effect of magnesium ion on cell-free synthesis of read-through protein of bacteriophage Qβ

This report describes the enhancing effect of magnesium ion on the synthesis of read-through protein of bacteriophage Qβ in a cell-free protein synthesizing system from E. coli . At 6 mM of magnesium acetate, the major product was coat protein. At 12 mM of magnesium, it was replaced by read-through protein. This enhanced synthesis was substituted by the addition of 0.25 mM of spermine or 1 mM of spermidine to 6 mM of magnesium. These results suggest that magnesium or combination of magnesium and polyamines causes leaky termination at the end of the coat protein cistron of Qβ-RNA.

Readinbg of the lysine codons in the MS 2 coat protein cistron during protein synthesis in vitro

The translation of the genetic information contained in the messenger RNA is based on the recognition between codon and anticodon. The first two positians of the codon are read by the anticodon strictly according to the rules of classic base pairing. Wowever, in the reading of the third codon position by the wobble nucleotide of the a~tic~dQ~ a greater degree of freedom must be postulated in order to allow for the discrepancy between the large number of codons in a degenerate code and the limited number of anticodons available for reading them. In his wobble hypothesis [I] Crick has laid down the restrictions which presumably govern the reading of the third codon position. Obviously, the reading of this position must be restricted in alI cases where there would otherwise be a mistake in the synthesized protein. For intance, in the phenyl~~ine~eu~~e codon group QJUU, UUC, UUA and UUG), an anticodon with Gin the wobble position cannot recognize the codons UUA and UUG because that would lead to the in~oduction of phenyl~an~e instead of leucine. We have introduced the term codon family to denote a group of four codons which all code for the same amino acid and we have asked ourselves if the rules of the wobble hypothesis apply as strictly here

Formation of ribosome-RNA initiation complexes with alfalfa mosaic virus RNA 4 and RNA 3.

RNA 4 of alfalfa mosaic virus (AMV) is a monocistronic messenger for the coat protein. We have determined the sequence of the 40 +/- 2 nucleotides in RNA 4 that were protected in the initiation complex formed with wheat germ 80 S ribosomes from digestion by T1 or pancreatic ribonucleases. The AUG coat protein initiation codon was near the middle of this protected region. We have found two ribosome-binding sites in RNA 3. The principal one, near the 5' end, is the initiation site for the major translation product, a 35,000 dalton protein. The second site binds ribosomes only weakly, at the beginning of the "silent" coat protein cistron, and is similar but not identical to the initiation site protection site is discussed.

Sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA.

The sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA has been determined. The sequence contains the entire coat protein cistron as well as regions to its left and right. Sequence characterization was by conventional methods for use with uniformly 32P labeled RNA complemented by newer methods for in vitro 5' and 3' 32P end-labeling of RNA and its subsequent rapid analysis. The noncoding region separating the coat protein cistron from the 3' terminus is 204 residues long and may be folded into a clover-leaf-type secondary structure. The distribution of termination codons to the left of the coat protein cistron suggests that the end of the adjacent cistron is separated from the beginning of the coat protein cistron by only two nucleotides. The subgenomic viral coat protein mRNA was isolated from infected tissue and shown to be capped. The nontranslated sequence separating the cap from the AUG initiation codon is 9 residues long and thus overlaps a portion of the adjacent cistron on the genome RNA.