Molecular cloning and nucleotide sequence of the 30K and the coat protein cistron of TMV (tomato strain) genome.

The cDNA copies of tobacco mosaic virus (TMV)-tomato strain (L) genome were cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2, 161-170. (1982)) and the sequence of 1,614 nucleotides at the 3' end was determined. The sequence encompasses the 30K and the coat protein cistron which are located in residues 685-1, 479 and 203-682 from the 3' end of the genome respectively. The close relationship between the tomato and the common strain was shown on the level of the nucleotide sequence. Highly homologous regions are found in the 3' non-coding region, the assembly origin and the 5' flanking region of the 30K protein cistron. The comparison of the deduced amino acid sequence between the tomato and the common strain shows that the 30K protein is composed of the conserved N-terminal four-fifth and the highly divergent region near the C-terminus.     

RNA 2 of tobacco rattle virus strain TCM encodes an unexpected gene.

The sequence of the 3'-terminal 1210 nucleotides of RNA 1 and the complete sequence of 3389 nucleotides of RNA 2 of tobacco rattle virus (TRV) strain TCM has been deduced. The sequence of the 3'-terminal 1099 nucleotides of RNAs 1 and 2 was found to be identical. Thus the genome of this TRV strain is partially diploid, encoding a 16K protein in both RNA 1 and RNA 2. The sequence that is unique to RNA 2 contains two open reading frames: the coat protein cistron and a cistron for a 29.1K protein, which shows no homology with the RNA 1 encoded 28.8K protein. cDNA probes corresponding to these two open reading frames cross-hybridized to pea early-browning virus RNA 2, but not to RNA 2 of five other tobraviruses tested.     

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

Summary 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.     

The functional and structural homology of ribosomal protein S7 of E. coli strains K and MRE600

Summary The 30S ribosomal protein S7 purified from E. coli MRE600 displaces specifically and stoichiometrically the endogenous K-S7 protein when it is added to a reconstitution system containing total K strain 30S protein and 16S RNA. The S7 proteins from the two strains have been shown to contain a group of common trypic peptides and to crossreact immunologically. Therefore, the 30S ribosomal protein S7 from E. coli K strain and MRE600 are functionally and structurally homologous despite differences in amino acid composition, molecular weight and electrophoretic mobility.     

Non-Continuous Translation of Coat Protein and Ribonucleic Acid Polymerase Cistrons in MS2 Bacteriophage Ribonucleic Acid

In an MS2 phage ribonucleic acid (RNA)-directed in vitro protein-synthesizing system, the coat protein cistron and the adjacent RNA polymerase cistron are translated non-continuously. The ribosomes which have completed the synthesis of coat protein dissociate from the MS2 RNA and do not read through the intercistronic gap. Translation of the adjacent RNA polymerase cistron requires ribosomes other than those translating the coat protein cistron.     

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.     

Studies on the bacteriophage MS2. XVI. The termination signal of the A protein cistron

The RNA of bacteriophage MS2 codes for three viral proteins: the coat protein, the A protein and the replicase. Upon infection of various amber suppressor strains of Escherichia coli, we found a fourth viral protein, the synthesis of which was specifically dependent on the presence of an amber suppressor gene. It is shown that this polypeptide is formed by reading through the natural termination signal of the A protein cistron. This cistron therefore terminates with the nonsense codon UAG. The observed prolongation accounts for the addition of some 30 amino acids. Unlike the normal A protein, the longer polypeptide is probably not incorporated into mature phage particles.     

Characterization of methylated neutral amino acids from Escherichia coli ribosomes.

The methylated neutral amino acids from both 30S and 50S ribosomal subunits of an Escherichia coli K strain were characterized. The 50S ribosomal subunit contains three methylated neutral amino acids: N-monomethylalanine, N-monomethylmethionine, and an as yet unidentified methylated amino acid found in protein L11. Both N-monomethylalanine and N-monomethylmethionine were found in protein L33. The amount of N-monomethylmethionine in this protein, however, is variable but not more than 0.25 molecules per protein. Thus protein L33 from this E. coli K strain has heterogeneity in its N-terminal amino acid and can start with either N-monomethylalanine or N-monomethylmethionine. The N-monomethylmethionine residue was not derived from the reduction of N-formylmethionine in the protein. The 30S ribosomal subunit contains only one methylated neutral amino acid: N-monomethylalanine.     

Nucleotide sequence of the tobacco mosaic virus (tomato strain) genome and comparison with the common strain genome

The sequence of about 4,500 nucleotides of the internal part of tobacco mosaic virus (TMV)-tomato strain (L) RNA has been newly determined using cloned cDNAs. Together with the previously determined partial sequences at both ends, the entire sequence of the 6,384 nucleotide genome has been completed. The 130K (1,115 amino acids), 180K (1,615 amino acids), 30K (263 amino acids) and coat protein (158 amino acids) cistrons are located at residues 72-3442, 72-4922, 4906-5700, and 5703-6182 on the genome, respectively. Sequence polymorphism was not observed except for heterogeneity in the length of the A cluster near the 3' end. The homology of the nucleotide sequences of TMV-L and TMV-vulgare, a common strain, is about 80% on average. Remarkable differences between them were found in a part of the N-terminal portion of the 130K/180K protein and the C-terminal portion of the 30K protein. A new method for cDNA cloning was developed by which the cDNA of the 5'-terminus of viral RNA can be cloned efficiently.     

The nucleotide sequence of tobacco vein mottling virus RNA.

The nucleotide sequence of the RNA of tobacco vein mottling virus, a member of the potyvirus group, was determined. The RNA was found to be 9471 residues in length, excluding a 3'-terminal poly(A) tail. The first three AUG codons from the 5'-terminus were followed by in-frame termination codons. The fourth, at position 206, was the beginning of an open reading frame of 9015 residues which could encode a polyprotein of 340 kDa. No other long open reading frames were present in the sequence or its complement. This AUG was present in the sequence AGGCCAUG, which is similar to the consensus initiation sequence shared by most eukaryotic mRNAs. The chemically-determined amino acid compositions of the helper component and coat proteins were similar to those predicted from the nucleotide sequence. Amino acid sequencing of coat protein from which an amino-terminal peptide had been removed allowed exact location of the coat protein cistron. A consensus sequence of V-(R or K)-F-Q was found on the N-terminal sides of proposed cleavage sites for proteolytic processing of the polyprotein.     

Two modes of amber codon read-through in vitro

Read-through translation of bacteriophage R17 amB2 coat cistron carrying an amber mutation at the seventh codon was studied in vitro using the crude cell extract (S30) derived from an Escherichia coli nonsuppressor strain. Despite the presence of termination factors as well as ribosome-releasing factor (RRF) which prevent the read-through translation [M. Ryoji, J. W. Karpen, and A. Kaji (1981) J. Biol. Chem. 256, 5798-5801], synthesis of coat-like protein still persists at a low level in this system. Characterization of this protein by peptide fingerprinting and amino acid sequencing was performed to reexamine the generally accepted notion that it is produced by amino acid misinsertion to the amber mutation codon. The results indicated, however, that the major population of this coat-like protein is produced as a result of reinitiation of translation from the eighth codon. Read-through by amino acid misinsertion in this system becomes predominant only when the Mg2+ concentration is higher than 16 mM.     

Nucleotide sequence of beet western yellows virus RNA.

The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellow virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southern bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cistron may be translated by in-frame readthrough of the coat protein cistron amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).     

Binding of mammalian ribosomes to MS2 phage RNA reveals an overlapping gene encoding a lysis function.

The main binding site for mammalian ribosomes on the single-stranded RNA of bacteriophage MS2 is located nine tenths of the way through the coat protein gene. Translation initiated at an AUG triplet in the +1 frame yields a 75 amino acid polypeptide which terminates within the synthetase gene at a UAA codon, also in the +1 frame. Partial amino acid sequence analysis of the product synthesized in relatively large amounts by mammalian ribosomes confirms this assignment of the overlapping cistron. The same protein is made in an E. coli cell-free system, but only in very small amounts. Analysis of the translation products directed by RNA from op3, a UGA nonsense mutant of phage f2, identifies the overlapping cistron as a lysis gene. In this paper we show that the op3 mutation is a C yield U transition occurring in the second codon of the synthetase cistron, which explains the lowered production of phage replicase (as well as lack of lysis) upon op3 infection of nonpermissive cells. We discuss the properties of the overlapping gene in relation to its lysis function, recognition of the lysis initiator region by E. coli versus eucaryotic ribosomes and op3 as a ribosome binding site mutant for the f2 synthetase cistron.     

The Nucleotide Sequence of the 3'-Terminal Region of Clover Yellow Vein Potyvirus Isolated from Calanthe sp.

The sequence of 1399 nucleotides from the 3'region of the RNA of clover yellow vein potyvirus (CIYVV-C) isolated from Calanthe sp. was determined. The capsid protein cistron was composed of 915 nucleotides, and corresponded to a region encoding 305 amino acids with a calculated Mr of 34900: the adjacent 3'non-coding region was 74 nucleotides long. The length of the capsid protein gene of CIYVV-C is longer, but the 3'non-coding region is shorter than those of other CIYVV strains (CIYVV-30.CIYVV-NZ and CIYVV-B). The nucleotide sequence of the capsid protein gene and the 3'non-coding region of CIYVV-C RNA showed significantly higher homology with those of other CIYVV strains. These data suggest that the CIYVV-C capsid gene arose from a frame-shift mutation of the capsid gene of another CIYVV strain. or vice versa. CIYVV-C has a closer affinity to other CIYVV strains and to bean yellow mosaic virus than to the other potyviruses.     

Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer

The complete nucleotide sequence was determined for the putative RNA polymerase (183K protein) gene of tobacco mosaic virus (TMV) OM strain, which differed from the related strain, vulgare, by 51 positions in its nucleotide sequence and 6 residues in its amino acid sequence. Three segments of this 183K protein, each containing the sequence motif of methyltransferase (M), helicase (H), or RNA-dependent RNA polymerase (P), were expressed in Escherichia coli as fusion proteins with hexahistidine tags, and domain-specific antibodies were raised against purified His-tagged M and P polypeptides. By immunoaffinity purification, a template-specific RNA-dependent RNA polymerase containing a heterodimer of the full-length 183K and 126K (an amino-terminal-proximal portion of the 183K protein) viral proteins was isolated. We propose that the TMV RNA polymerase for minus-strand RNA synthesis is composed of one molecule each of the 183- and 126-kDa proteins, possibly together with two or more host proteins.     

Location of the cistron of the tobacco mosaic virus coat protein.

Treatment of tobacco mosaic virus (TMV) RNA with T1 RNase under mild conditions cuts the RNA molecule into a large number of fragments, only a few of which may be specifically recognized by disks of TMV protein. It has been shown elsewhere that these specifically recognized RNA fragments are a part of the coat protein cistron, the portion coding for amino acids 95 to 129 of the coat protein. It is reported that different size classes of partially uncoated virus particles were prepared by limited reconstitution between TMV RNA and protein or by partial stripping of intact virus with DMSO. Both procedures produce nucleoprotein rods in which the 5'-terminal portion of the RNA is encapsidated and the 3'-terminal region is free. The free and the encapsidated portions of the RNA were each tested for the ability to give rise to the aforesaid specifically recognized fragments of the coat protein cistron upon partial T1 RNase digestion. It was found that only the 3'-terminal third of the virus particle need to be uncoated in order to expose the portion of the RNA molecule from which these fragments are derived. We conclude, therefore, that the coat protein cistron is situated upon the 3'-terminal third of the RNA chain, i.e. within 2000 nucleotides of the 3'-end.     

Analysis of the genome structure of tobacco rattle virus strain PSG.

The sequence of the 3'-terminal 2077 nucleotides of genomic RNA 1 and the complete sequence of genomic RNA 2 of tobacco rattle virus (TRV, strain PSG) has been deduced. RNA 2 (1905 nucleotides) contains a single open reading frame for the viral coat protein (209 amino acids), flanked by 5'- and 3'-noncoding regions of 570 and 708 nucleotides, respectively. A subgenomic RNA (RNA 4) was found to lack the 5'-terminal 474 nucleotides of RNA 2 and is the putative messenger for coat protein. The deduced RNA 1 sequence contains the 3'-terminal part of a reading frame that probably corresponds to the TRV 170K protein and reading frames for a 29K protein and a 16K protein. Proteins encoded by the first two reading frames show significant amino acid sequence homology with corresponding proteins encoded by tobacco mosaic virus. Subgenomic RNAs 3 (1.6 kb) and 5 (0.7 kb) were identified as the putative messengers for the 29K and 16K proteins, respectively. At their 3'-termini all PSG-RNAs have an identical sequence of 497 nucleotides; at the 5'-termini homology is limited to 5 to 10 bases.     

Ribonucleic Acid and Protein Synthesis in a Mutant of Bacillus subtilis Defective in Potassium Retention

A mutant of Bacillus subtilis 168 (strain 168 KL), which had lost its normal capacity to accumulate K(+), was used to explore the interrelationship between protein and ribonucleic acid (RNA) synthesis. In contrast to the wild type, the growth rate of strain 168 KL was markedly dependent on the K(+) concentration in the medium. K(+) uptake in the mutant strain was identical to that in the parent, but the mutant was unable to retain and accumulate K(+). Protein synthesis was markedly dependent on the K(+) concentration in the medium, whereas RNA synthesis was relatively unaffected by changes in the level of K(+). Most of the RNA synthesized during K(+) depletion was ribosomal RNA; it appeared in crude extracts in the form of ribonucleoproteins particles with sedimentation values between 4S and 30S. These particles were converted into mature ribosomes when growth was allowed to resume by the addition of K(+). Simultaneous synthesis of RNA and protein was necessary for the quantitative conversion of the ribonucleoprotein particles into ribosomes. During recovery from K(+) depletion, ribosomal protein was synthesized in preference to the other proteins of the cell.     

Observations concerning the sequence of two additional specifically encapsidated RNA fragments originating from the tobacco-mosaic-virus coat-protein cistron.

The incubation of 25-S tobacco mosaic virus (TMV) protein with a mixture of RNA fragments produced by partial T1 RNase digestion of TMV RNA results in the encapsidation of only a few species of RNA. In addition to the most predominant species, fragment 1, whose sequence has been described in the prededing paper, two other species, fragment 41 and fragment 21 are coated by the protein. These two RNA fragments were purified by polyacrylamide gel electrophoresis and subjected to total digestion with pancreatic and T1 RNase. The oligonucleotides were separated by paper electrophoresis and characterized insofar as possible by digestion with the complementary ribonuclease. From the amino acid coding capacity of the oligonucleotides liberated from fragments 41 and 21 by T1 RNase digestion, it appears that these two fragments, like fragment 1, are derived from the coat protein cistron. They are situated immediately prior to fragment 1 and, together with this fragment, consitute a continuous stretch of 232 nucleotides of the cistron which codes for animo acids 53 to 130 of the coat protein. The order of the fragments in the sequence is 21-41-1. A possible model for the secondary structure of this portion of the sequence is proposed.     

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.