Article

Observations Concerning the Sequence of Two Additional Specifically Encapsidated RNA Fragments Originating from the Tobacco‐Mosaic‐Virus Coat‐Protein Cistron

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Abstract

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.

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Article
A study of the synthesis of virus-specific proteins is closely related to the regulatory aspect of gene expression. This chapter focuses on the peculiarities of the translation of plant virus mRNAs and some mechanisms controlling the process. General principles of mRNA replication and expression with respect to viruses containing virion plus-strand RNA, minus-strand RNA, double-stranded RNA (dsRNA), and DNA are discussed in the chapter. Functions encoded in the genome of RNA-containing plant viruses are performed by the corresponding gene products-virus-specific (i.e., virus-coded) proteins. Aside from the virus-specific proteins, virus multiplication may be either positively or negatively affected by the products (proteins or possibly RNAs) coded for by the host cell genome, synthesis of which is induced by virus infection (so-called virus-induced products). An example of a virus-induced protein in the virus-animal cell system is interferon. Plant virologists have realized that the coat proteins of many viruses are synthesized in excess with respect to other virus-coded products. Ion concentration in cell-free extracts is an important regulatory factor in the translation of RNAs of viruses. There is some evidence that ionic concentration may influence the process of plant virus RNA translation in vitro.
Article
Recognition of the unique internal assembly origin on tobacco mosaic virus (TMV) RNA by the disk aggregate of the viral coat protein probably involves an extended region of the RNA (larger than that coated by a single disk) folded into a specific conformation. A secondary structure model is proposed for the RNA preferentially coated by limiting amounts of coat protein disks on the basis of partial nuclease digestion data. Part of this sequence can form three symmetrically spaced hairpins with marginally stable base paired sequences at the tips of the stems. The pattern of progressive protection of the RNA from nuclease attack during assembly suggests that these three hairpins are successively coated by the first three disks to add. The spacing of these hairpins is identical to that of three hairpins in the pseudo assembly origin (part of the coat protein gene homologous to the assembly origin). In Ni 2519, a TMV mutant whose assembly is defective at high temperature because it can no longer discriminate between the true and pseudo assembly origins, a point mutation has occurred near the tip of the third metastably base paired stem of the true assembly origin which would disrupt its structure and alter one copy of a repeated heptanucleotide. This suggests an important role for the ordered and cooperative recognition of successive loops in determining the specificity of assembly.
Article
Tobacco mosaic virus (TMV) consists of rigid rod-like nucleoprotein particles containing about 5% RNA, which can be easily transmitted to a wide range of plant hosts by mechanical inoculation. A kilogram of infected tissue from the appropriate variety of tobacco can render several grams of purified TMV, and this high yield, along with the great stability of the purified virus, has made TMV a favored object of investigation over the years. TMV was, for example, the first virus to be purified, the first virus shown to contain RNA as genetic material, and the first virus to be reassembled in vitro from its protein and RNA components. It was the first virus shown to have its protein subunits disposed in a regular fashion, and recently, x-ray diffraction of oriented fibers of TMV virions and of crystals of TMV protein disks has extended the vision of the fine structure of much of the virion to a resolution of better than 3 Å. The extent of the progress in understanding TMV from a structural point of view may be measured by the number of detailed reviews devoted to this subject.
Article
A method is developed to study the periodic properties of nucleotide sequences allowing the favoured pattern of the repeating unit, as well as the length and localization of this periodic segment to be determined simultaneously. The degree of periodicity is evaluated calculating the probabilities for random occurrence of the maximal deviations of the nucleotide composition in each phase, making use of the binomial formula.The nucleotide sequence of the tobacco mosaic virus (TMV) RNA responsible for recognition of the homologous protein (“assembly origin”, AO) (Zimmern & Butler, 1977) was investigated in order to find periodic regions of primary structure which might be essential in the recognition process. As a result the most periodic segments of the AO consisting of 31 and 17 nucleotides corresponding to the schemes GAU or GA∗ have been found. However, the periodicities in these regions do not exceed that expected for random sequences. It can be considered as an evidence that in addition to peculiarities of primary structure, some other features such as RNA secondary or tertiary structure are essential in this interaction.For comparison the nucleotide sequences of the other fragments of TMV RNA as well as MS2 RNA, TYMV RNA, 16S rRNA and phage fd DNA were investigated by the same method.
Article
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.
Article
Assembly of nucleoprotein rods from tobacco mosaic virus (TMV) coat protein and poly(A) depends on the presence of 20S disks in a manner very similar to nucleation and growth of virions in reconstitution with TMV RNA. Products assembled with (A) approximately equal to 5000 appear to have the same buoyant density in CsCl, the same nucleotide/protein ratio and the same nuclease stability, as reconstituted and native TMV. Their rate of formation is very similar to the rate of reconstitution with TMV RNA when high-molecular-mass (A) approximately equal to 5000 is used, but becomes a function of chain length particularly with (A) less than or equal to 185. The composition of assembly products can be described sufficiently with the relation between number of capsid polypeptide monomers/particle, np, to the number of nucleotide residues/chain, nnt, of np = 1/3 (nnt + 50) with two important restrictions: (1) particles of less than four turns of helically arranged capsid subunits are unstable, and (2) particles with about 150 or less nucleotides per chain deviate in structure from mature virus and virus-like (= longer) assembly products. This is indicated by changes in both buoyant density in CsCl and optical properties, while 'dislocation' of the disk to the helical arrangement of capsid subunits ('helicalization') and nuclease stability already become established with chains as short as (A) approximately equal to 58 +/- 20. Consequently, we suggest that assembly proceeds through three distinct phases: (1) nucleation (resulting in helicalization) by interaction of nucleic acid with the first disk; (2) stabilization of the primary (unstable!) nucleation complex by addition of a second disk and formation of a four-turn virus-like and stable nucleoprotein helix, which is then fit for (3) elongation by addition of further disks. The question of what makes the TMV protein disk select specifically TMV RNA during virion assembly is discussed in some detail.
Article
The assembly origin of tobacco mosaic virus RNA contains three stable hairpin loops. Coat protein disks bind first to loop 1 (the 3' most) during virus assembly, but the whole region is coated in a concerted fashion even in conditions of limiting protein. It is shown by in vitropackaging assays using mutant in the assembly origin transcripts that rapid and specific assembly initiation occurs in the absence of loops 2 and 3, but is abolished on removal of loop 1. Deletion or alteration of the unpaired AAGAAGUCG sequence at the apex of loop 1 also abolishes rapid packaging; this sequence is therefore instrumental in disk binding. Alteration of this sequence to (A)9 leads to packaging at a very low rate (half time 12 hours) which is apparently non-sequence specific. Substitution of (CCG)3 evokes packaging with a half time of 3 hours, as compared to 15 seconds for the wild type assembly origin. These results suggest that the three-base G periodicity within this sequence element is an important feature in assembly nucleation.
Article
TMV assembly starts with a specific interaction between the assembly origin on the RNA and a disk aggregate of coat protein. The assembly origin is located in the 30K protein cistron for common and tomato strains of TMV and in the coat protein cistron for cowpea strain of TMV and for CGMMV. All the assembly origins have three essential structures: a long base-paired hairpin loop structure; a target sequence, GAPuGUUG, at the top of the hairpin loop structure; and a tract where every third base is a purine. The protein aggregate responsible for the initiation of TMV assembly is a 20S disk, a two-layered aggregate of 34 protein subunits. The two layers of a disk open apart onto the central hole and this structure may be critical for the disk to interact with the assembly origin on the RNA. The target sequence may bind specifically to this structure. Although only a low concentration of 20S disks exists in the usual assembly condition, one disk is enough to initiate TMV assembly. TMV elongation proceeds in two directions. Elongation to the 5'-end proceeds rapidly by preferential incorporation of protein subunits (or A protein) and in 5-7 min gives rise to 260 nm intermediate particles whose 5'-end is coated. A model of elongation toward the 5'-end is shown in Fig. 15. Protected RNAs from nuclease digestion during the assembly reaction produce a banding pattern on gels by electrophoresis. The banding pattern reflects features of the RNA rather than protein that are used in the assembly reaction, since the pattern was the same for assembly between TMV-RNA and CGMMV protein subunits as for assembly between TMV-RNA and TMV protein containing 20S aggregates. The 20S aggregate in the assembly solution has a helical structure with 39 protein subunits rather than the disk structure. Rapid addition of 20S helical aggregates to the top of the growing rod seems to be impossible because of its topological complexity. Elongation toward the 3'-end does not start for at least the first 4 min after initiation. It probably cannot begin until the 5'-end RNA tail disappears into the intermediate rod. Elongation toward the 3'-end favors 20S aggregates as the protein source and gives rise to the full-length rods in about 30 min after the initiation. There are no topological difficulties in adding 20S helical aggregates to the protruding RNA tail.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
THE optimum efficiency of protein biosynthesis depends on the availability of aminoacyl-transfer RNAs (tRNAs) in the neighbourhood of active polysomes, among other factors. The intracellular tRNA level is directly proportional to the composition of the amino acids incorporated into proteins being synthesised1. This continuous and selective adjustment takes place in prokaryotes and eukaryotes and in non-differentiated as well as in highly specialised cell systems2. We report here that such a proportionality takes place at the molecular level between the frequency of synoinomous codons in messenger RNAs (mRNAs) and the distribution of the corresponding isoacceptor tRNAs. A correlation was found for the alanine, glycine and serine codons from viral nucleic acids and eukaroytic iso-tRNA sets fractionated on reversed-phase chromatography as well as for the preponderant tRNAs involved in the translation of fibroin mRNA in the posterior silk gland of the silkworm Bombyx mori L.
Article
The products of in vitro reconstitution of brome mosaic virus (BMV) protein and RNA were studied using analytical sedimentation and polyacrylamide gel electrophoresis. The product formed depended on the protein concentration, even though the ratio of RNA to protein was constant (1:4). At a low ionic strength with protein at 0.2 g/liter, mostly 78 S particles (like native BMV) were made, whereas at 2 g/liter of protein, reconstitution proceeded erratically. When the ionic strength was decreased to various levels by dialysis after mixing RNA and protein at I = 1.0, the four RNAs were encapsidated at different rates according to species, with RNAs 4 and 3 at the highest rates. The same sequential encapsidation in order of increasing molecular weight occurred as the ratio of protein to RNA was increased. Some particles containing three copies of the smallest RNA were found. These observations show that BMV protein displays in vitro a differential affinity for the various BMV RNA species.
Article
This chapter describes the structure, functional design of the tobacco mosaic virus (TMV), protein aggregation, nucleation of assembly, rod elongation, selectivity for viral RNA, and general considerations. TMV is a very infectious high-yielding virus, whose properties have been highly optimized by evolution. It may therefore be possible to discern biological advantages in many of the features of its protein and RNA. Outside the plant host cell, the coat protein must ensure that the virion remains stable. Inside a susceptible plant cell, the RNA must interact with the host's biochemical machinery to ensure its own transcription and replication. TMV protein can form helical rods that are essentially the same as the protein part of the virus. Parts of TMV RNA sequence are being investigated, and sequences are available for the 71 nucleotides at the 3’-hydroxyl end and for several other oligonucleotides from within the RNA. Comparisons between these and the stronger binding regions, together with the electron density maps, may make it possible to understand the protein-nucleic acid interaction. While, it is not yet clear how generally untranslated mRNA occurs in eukaryote cells, possibly as a mechanism to allow a rapid switching one of the synthesis of specific proteins, TMV RNA may find a new experimental use in investigation of the mechanisms and control of eukaryotic protein synthesis.
Article
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 cis-tron 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.
Article
The study described here concerns the proteins, synthesized as a result of tobacco mosaic virus (TMV) multiplication in tobacco protoplasts and in cowpea protoplasts. The identification of proteins involved in the TMV infection, for instance in the virus RNA replication, helps to elucidate the infection process in the plant cell. Not only virus coded proteins, but possibly also host coded proteins may play a part in the TMV multiplication.Research on proteins encoded by the TMV RNA, carried out in cell-free protein synthesizing systems, has revealed that five polypeptides are synthesized under the direction of TMV (subgenomic) mRNAs (see table 1.2., chapter L). Whether the polypeptides, synthesized invitro with TMV RNA as messenger, are of functional significance for the TMV infection may only be determined by means of investigating TMV infected leaves and protoplasts.The TMV multiplication runs synchronously in all protoplasts that are infected. Therefore, proteins synthesized in small amounts upon infection, may be thus detected.The search for proteins sythesized in protoplasts as a result of TMV infection has long been hindered by the fact that various factors in the cultivation of the tobacco plants may adversely influence the quality of the protoplasts. The cultivation of the tobacco plants: Nicotiana Tabacum cv. L. Samsun, Samsun NN and Xanthi nc, could be standardized however, as described in chapter 2. When the tobacco plants were cultivated in this way, at least 50 % of the tobacco protoplasts could be infected with TMV and 70 % or more of the protoplasts survived the subsequent incubation period of 36 hours. This could be achieved every time the protoplasts were isolated. The intensity and quality of the light, the way of watering, the age of the tobacco plants and of the leaf, from which the protoplasts are isolated, among others, appeared to affect the quality of the protoplasts (chapter 3.).The proteins, synthesized upon TMV infection, have to be distinguished among a great variety of host proteins. For this reason it is important to determine the incorporation of radioactive amino acids into protein synthesized as a result of TMV multiplication, in comparison with the incorporation into host proteins that are formed independently from the virus infection. Therefore the specific activity of TMV coat protein (cpm/mg protein) and of the proteins of the 27,000 x g supernatant fraction, synthesized in infected tobacco protoplasts were compared. It appeared that the specific activity of TMV coat protein was at least four times higher than of the proteins in the 27,000 x g supernatant (chapter 4.).The proteins synthesized as a result of TMV multiplication were studied not only in tobacco protoplasts, but also in protoplasts from the primary leaves of cowpea ( Vigna unguiculata (L.) Walp. var. 'Blackeye Early Ramshorn'). The method used for the infection of tobacco protoplasts with TMV was not suitable for the infection of cowpea protoplasts with TMV. Best results were obtained when both protoplasts and virus were incubated in the presence of poly-D-lysine, for 7.5 min. before infection. The protoplasts were pre-incubated in 0.1 M potassium phosphate buffer (pH 5.4) at 0°C, at a concentration of 4 x 10 5 protoplasts/mI and 0.75 μg poly-D-lysine/ml. TMV was pre-incubated in the same buffer at room temperature at a concentration of 2 μg TMV/mI and 2 μg poly-D-lysine/ml. During infection the cowpea protoplasts were incubated together with TMV and poly-D-lysine in a concentration of 2 x 10 5 protoplasts/ml, 1 μg TMV/ml and 1 μg poly-D-lysine/ml, for 7.5 min, in the buffer mentioned above at 0°C. In this way 50 to 70 % of the cowpea protoplasts could be infected with TMV.The course of TMV synthesis in cowpea protoplasts was comparable with that in tobacco protoplasts. The TMV multiplication in cowpea protoplasts was preceeded, however, by a period of 16 hours, during which the increase of TMV is slight, while the TMV multiplication in tobacco protoplasts was preceeded by a lag period of 8 hours. A possible explanation is that a much smaller amount of TMV particles penetrates into cowpea protoplasts during inoculation and/or starts to multiply than is the case in tobacco protoplasts (chapter 5.).The proteins of TMV infected and mock-infected protoplasts were analysed therupon by means of SDS-polyacrylamide slabgel electrophoresis and the polypeptide patterns were visualized by autoradiography.Ten polypeptides were distinguished, which are synthesized as a result of TMV multiplication in polypeptide patterns of proteins from infected tobacco protoplasts. The molecular weights were estimated to be 260,000, 240,000, 170,000, 116,500, 96,000, 90,000, 82,000, 72,000, 30,000 and 17,500 (coat protein). Polypeptides of similar molecular weight were absent or were present to much less extent in polypeptide patterns of proteins from mock-infected tobacco protoplasts. Many polypeptides were observed for reason that the detection capacity was improved by means of subcellular fractionation of the protoplast homogenates.The polypeptides of molecular weight 170,000, 116,500, 72,000 and coat protein were present in the 31,000 x g supernatant fraction and the pellet fractions as well. The polypeptide of molecular weight of 30,000 was present exclusively in the pellet fractions. The other polypeptides were observed exclusively in polypeptide patterns of protein of the 31,000 x g supernatant fraction (see table 6. l., chapter 6.).Eight polypeptides were observed, which were synthesized as a result of TMV multiplication in cowpea protoplasts. The molecular weights of the polypeptides were approximately 150,000, 116,500, 86,000, 72,000, 17,500 (coat protein), 16,000,14,000 and 10,000. Polypeptides of similar molecular weight were absent or present on a far less extent in polypeptide patterns of proteins from mockinfected cowpea protoplasts.The polypeptides of molecular weight 116,500, 72,000 and coat protein were present in the 3 1,000 xg pellet and 3 1,000 xg supernatant. The other polyeptides were present exclusively in the 3 1,000 xg supernatant (table 7. l., chapter 7.).It was assumed that the TMV coded polypeptides are similar in different hosts and, on the other hand, that the host polypeptides, synthesized upon TMV infection differ from host to host. When the TMV specific polypeptides, synthesized in infected tobacco protoplasts were compared with the specific polypeptides synthesized in TMV infected cowpea protoplasts, it appeared that only the polypeptides of molecular weight 116,500, 72,000 and coat protein are of similar size in both hosts (table 7.2., chapter 7). This is an indication that not only the polypeptide of 116,500 daltons and coat protein are TMV coded polypeptides, but that also the polypeptide of 72,000 daltons is encoded in the TMV RNA. It has not been reported that a polypeptide of this size is observed when TMV RNAs are translated in cell-free protein synthesizing systems.A polypeptide of 170,000 daltons is synthesized in vitro under the direction of the TMV RNA. It appeared that the polypeptide synthesized in TMV infected tobacco leaves, has a slightly less electrophoretic mobility than the product of 170,000 daltons synthesized in vitro from TMV RNA as messenger. A polypeptide of similar electrophoretic mobility was present to a lesser extent in mockinfected tobacco protoplasts. Furthermore, a polypeptide of 170,000 daltons was not observed in TMV infected cowpea protoplasts. For these reasons it is likely, that the polypeptide of 170,000 daltons, synthesized in TMV infected tobacco protoplasts, is encoded in the genome of tobacco or is encoded in the TMV RNA, but then the polypeptide has no functional significance in the TMV multiplication process.Further the polypeptide of 30,000 was observed only in TMV infected tobacco protoplasts, whereas a polypeptide of similar molecular weight was shown to be synthesized in vitro from a TMV subgenomic mRNA. The polypeptide of 30,000 daltons was detected exclusively in the polypeptide patterns of protein from the pellet fractions of TMV infected tobacco protoplasts. Polypeptide patterns of protein from corresponding fractions of cowpea protoplasts had a predominant, grey background. Due to this the polypeptide of 30,000 daltons may not be distinguished in TMV infected cowpea protoplasts, whereas the polypeptide of 30,000 daltons synthesized in TMV infected tobacco protoplasts can in fact be a polypeptide coded by TMV RNA. The other polypeptides synthesized in infected tobacco protoplasts or cowpea protoplasts as a result of TMV multiplication are presumably synthesized under the genome of tobacco or cowpea respectively.Finally, it was attempted to examine in what way the polypeptides of 116,500 and 72,000 are involved in the TMV infection process. Both polypeptides were shown to be present in the 31,000 x g pellet of TMV infected tobacco and cowpea protoplasts. It was studied whether virus specific polypeptides of similar molecular weight can be observed in RNA-dependent RNA polymerase preparations isolated from the 31,000 x g pellet fraction of cowpea leaves infected with the cowpea strain of TMV (C-TMV). The RNA-dependent RNA polymerase preparations were isolated by extraction of the 31,000 x g pellet fraction and were further purified by means of subsequent DEAE-BioGel column chromatography and glycerol gradient centrifugation. The purification procedure used was the same procedure as described for the isolation of RNA-dependent RNA polymerase from cowpea leaves infected with cowpea mosiac virus (CPMV).Four specific polypeptides of molecular weight of 98,000, 90,000, 72,000 and 46,000 were distinguished in RNA-dependent RNA polymerase preparations from C-TMV infected cowpea leaves, after glycerol gradient purifications. A polypeptide of molecular weight 116,500 was not observed. Polypeptides of molecular weights 72,000 and 46,000 were not found and those of molecular weights 98,000 and 90,000 were distinguished to a less extent in polypeptide patterns of preparations isolated in exactly the same way from mock-inoculated cowpea leaves.RNA-dependent RNA polymerase activity was also observed in preparations isolated from mock-inoculated cowpea leaves. The specific activity (cpm/mg protein) of the preparation from mock-inoculated leaves was one sixth of the specific activity of the RNA-dependent RNA polymerase preparations from CTMV infected cowpea leaves. The RNA-dependent RNA polymerase activity in C-TMV infected cowpea leaves might therefore be attributed to the increase of one or several polypeptides, present already before inoculation. Since it was thought that the polypeptide of 72,000 daltons is a TMV coded polypeptide, it was examined which specific polypeptides are present in RNA-dependent RNA polymerase preparations isolated in a similar way from CPMV infected cowpea leaves. It appeared, that in addition to CPMV specific polypeptides, the polypeptides of molecular weight 98,000 and 90,000 were also observed in RNAdependent RNA polymerase preparations from CPMV infected leaves. The polypeptides of 72,000 and 46,000 daltons were distinguished only in preparations isolated from C-TMV infected cowpea leaves. These results suggest that the polypeptide of 72,000 daltons in involved is the synthesis of TMV RNA (chapter 8.).
Article
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 m7G 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.
Article
Complementary DNA (cDNA) copies of the common (OM) and Cowpea (Cc) strains of tobacco mosaic virus (TMV) RNA polyadenylated in vitro have been synthesized with AMV reverse transcriptase and oligo (dT)10 as primer. The cDNA was converted to a double-stranded form by E. coli DNA polymerase I followed by S1 nuclease digestion. The double-stranded cDNA copies were cloned in pBR322 at the PstI site using the oligo(dC)-oligo(dG) tailing method. With the common strain, several clones containing inserts covering different parts of genomic RNA were selected using partially reconstituted RNA and partially stripped virus RNA as hybridization probes. Restriction mapping of three clones and their overlaps showed that cloned sequences covered about 4000 nucleotides of the common strain RNA from the 3' end. With the Cc strain, clones containing the 3' portion were selected by Southern hybridization using coat protein mRNA isolated from short particles as a probe. One cloned recombinant plasmid was found by restriction analysis and R-loop mapping to carry about a 1700 nucleotide sequence of Cc strain RNA from the 3' end. A restriction map of the OM strain was very similar to the map of the vulgare strain, as predicted from its nucleotide sequence, but completely different from that of Cc strain.
Article
It was suggested in an accompanying paper [Taliansky et al. (1982b), Virology, 117, 000-000] that two reconstitution initiation sites (RIS I and RIS II) were functionally active in RNA of the temperature-sensitive (ts) TMV mutant Ni2519 upon reassembly at a nonpermissive temperature; initiation of reassembly that started at two sites on the Ni2519 RNA molecule resulted in defective (ribonuclease-sensitive) virus particle (DVP) formation. RIS(s) was(were) defined as a segment(s) of Ni2519 RNA protected from the action of ribonuclease T1 by the coat protein within an incomplete nucleoprotein complex (I-NPC) formed upon limited TMV reassembly at nonpermissive (33 degrees and permissive (24 degrees) temperatures. Tl-resistant oligonucleotides protected within I-NPC were finger-printed, sequenced, and assigned to specific regions on the Ni2519 RNA molecule. It was shown that only RIS I was operative on Ni2519 RNA at 24 degrees as well as on A14 (a temperature-resistant strain, the wild type for Ni2519) RNA at both 24 and 33 degrees ; RIS I corresponded to the so-called Oa (origin of assembly) of a common TMV strain previously studied and was located at a distance of about 800 nucleotides from the 3'-end of the RNA molecule. In contrast, two RISs were revealed on Ni2519 RNA assembled at 33 degrees ; of the two RISs operative in this case the first one is identified as RIS I, while the second (RIS II) is located within 300-520 nucleotides from the 3'-end, i.e., within the coat protein gene. The latter corresponds to the so-called SERF (specifically encapsidated RNA fragment) of the RNA of common TMV.
Article
Double-stranded cDNA copies of cucumber green mottle mosaic virus (watermelon strain, CGMMV-W) RNA polyadenylated in vitro were cloned into the pBR322 at the PstI site. The sequence of 1071 nucleotides from the Tend of the genomic RNA was determined using two recombinant plasmids and the genomic RNA. The coat protein cistron was located in residues 176-661 from the 3' end. The coat protein was composed of 160 amino acid residues with the molecular weight of 17,261. The 3' noncoding region of the CGMMVW genome was 175 nucleotides long and highly homologous to that of the common strain of TMV. The assembly origin of reconstitution is positioned within the coat protein cistron as predicted previously. In the 5' flanking region of the coat protein cistron a long open frame, probably of 30K protein, was found. The predicted 30K and the coat protein cistron would overlap each other as is the case of the cowpea strain of TMV.
Chapter
Tobacco mosaic virus (TMV) has become a classical object for studies on the structure and assembly of viruses. Shortly after the first purification of virus by Stanley (1935), structural studies were begun using the methods of biochemistry (Bawden and Pirie, 1937) and X-ray diffraction (Bernal and Fankuchen, 1941). The difficulties encountered in elucidating a structure of this size led to many developments in both techniques and instrumentation which enabled the structure to be determined at steadily increasing resolution. Since the virus has a helical structure (Watson, 1954) and forms highly ordered gels rather than single crystals, the three-dimensional structure must be determined by deconvolution from two-dimensional diffraction patterns of the virus gels which are azimuthally disordered. Notwithstanding the difficulties imposed by this, the virus structure has been solved to a resolution approaching 0.4 nm (Stubbs et al., 1977). In the absence of RNA, the protein will form true crystals of one of its aggregates (the disk; see Section II.B.1) whose structure has been solved to a resolution beyond 3 nm which allowed an atomic model to be built (Bloomer et al., 1978).
Chapter
Messenger RNA has the central role in gene expression of conveying the information for making polypeptides from DNA (or, in some cases, RNA) to the ribosomes. Although the concept of mRNA as the direct template for polypeptide synthesis came to be widely accepted following the proposals of Jacob and Monod (1961), it is only in recent years that mRNA has become a well-defined biochemical entity. A number of individual cellular mRNAs have now been substantially purified and shown to direct the synthesis of the expected polypeptides in cell-free systems; nucleotide sequence analysis of one phage RNA that functions as a messenger has been carried to completion, and a good start has been made in dissecting the structure of cellular mRNAs. Whereas a messenger was once pictured simply as a string of nucleotides containing the triplet codons for aminoacyl-tRNAs, we now realize that many mRNAs contain significant amounts of untranslated nucleotide sequences, that ribosomes (and probably some proteins) recognize specific attachment sites in mRNAs, that mRNAs have elements of secondary and tertiary structure, and that many mRNAs are modified after transcription in novel ways.
Chapter
The first natural homodisperse polyribonucleotides to become available for chemical characterization were the virion RNAs of plant viruses and certain animal viruses. The finding that the infectivity of many viruses, first of tobacco mosaic virus, was a property of their RNAs (Fraenkel-Conrat, 1956; Gierer and Schramm, 1956; Fraenkel-Conrat et al., 1957; Colter et al., 1957), and that these thus represented the first pure genes as well as mRNAs to become available, made studies of their nucleotide sequences obviously of great interest. The later and gradual realization that the virions of many other virus families, e. g., the rhabdo-, myxo-, paramyxo- and reoviridae, carried minus-strand or double-stranded RNAs which had to be transcribed before they could serve as messenger did not diminish the intrinsic importance and interest in their nucleotide sequence.
Chapter
Tobacco mosaic virus (TMV), a positive-strand RNA virus, provides a unique perspective on nucleic acid structure, in that it has been for some years the only macromolecular assembly in which the structure of a ribonucleic acid interacting with a protein has been visualized in molecular detail, and one of the very few showing protein-nucleic acid interactions at all. Crystallographers have determined the structures of a number of spherical RNA viruses (for references see Liljas, 1986; Stubbs, 1989); but in those structures so far determined, the nucleic acid is not seen in the electron density maps, because it does not conform to the icosahedral symmetry of the coat proteins. TMV, by contrast, is a helical virus, and its RNA is well ordered, with the same symmetry as the protein. It is the type member of the tobamovirus group, rod-shaped viruses 3000 Å long and 180 Å in diameter, with a central hole of diameter 40 Å. Approximately 2130 identical protein subunits of molecular weight 17500 form a right-handed helix of pitch 23Å with 49 subunits in 3 turns. A single strand of RNA follows the basic helix between the protein subunits at a radius of 40 Å, with three nucleotides bound to each protein subunit. An overall view of part of the viral rod is shown in Figure 5.1.
Article
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.
Article
When 25-S tobacco mosaic virus (TMV) protein aggregate and TMV RNA, which has been partially digested by T1 RNase, are mixed under conditions suitable for reconstitution, only a few RNA fragments are encapsidated. These fragments were isolated and purified by polyacrylamide gel electrophoresis. The sequence of the three main fragments, the longest of which (fragment 1) was estimated to contain 103 nucleotides, has been determined. The two smaller fragments are portions of the longer chain produced by an additional specific scission. Because of the great affinity of 25-S TMV protein for this nucleotide sequence, it will be referred to as the "specifically encapsidated RNA fragment". The occurrence of a "hidden break" in the sequence has been demonstrated: fragment 1, purified by electrophoresis on a polyacrylamide gel without 8 M urea, gives rise upon further electroporesis in the presence of urea to two new bands corresponding to the two halves of the molecule. A stable hair-pin secondary structure has been derived from the base sequence which can account for the specificity of action of the enzyme. Because of its properties, we have suggested elsewhere that the sequence of fragment 1 might correspond to the disk recognition site for reconstitution, which is known to be located at the 5' end of the intact RNA. But experiments with TMV RNA whose 5'-OH end has been radioactively phosphorylated with polynucleotide kinase show that this is not the case. Analysis of the amino acid coding capacity of the fragment has instead revealed that fragment 1 is a portion of the TMV coat protein cistron.
Article
Nucleotides from a ribonuclease digest of 32P-labelled 5 † ribosomal RNA of Escherichia coli have been studied by two-dimensional ionophoresis on modified celluloses. All the nucleotides from ribonuclease T1 and pancreatic ribonuclease digests were separated and their structure was determined. The yields of the smaller nucleotides were reproducible and greater than 70%. 5 s RNA consists of about 115 nucleotides of which pUG is 5′-terminal and GCAUOH is 3′-terminal.†The low molecular weight ribosomal RNA was conveniently termed "5s" RNA by Rosset et al. (1964) to distinguish it from "4s" or transfer RNA. This terminology will be used throughout this paper, although it is not meant to imply that the value of S is exactly 5·0.
Article
The binding of a few molecules [1-6] of RNA bacteriophage coat protein to 1 molecule of RNA represses in vitro translation of the RNA synthetase cistron. Digestion of the complex, R17 coat protein-R17 RNA, by T1 RNase yields an RNA fragment bound to the coat protein. The nucleotide sequence of this fragment (59 residues) reveals that it contains the punctuation signal between the coat protein and RNA synthetase cistrons, suggesting that this is the site on the RNA where the coat protein acts as a translational repressor.
Article
There are two major changes. First, instead of assigning free energies to each base pair, we will consider the base pairs two at a time.
Article
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.
Article
Bacteriophage R17 RNA was labelled with (32)P and was subjected to partial digestion with ribonuclease T(1). The products were fractionated by ionophoresis on polyacrylamide gel. Two fragments were purified and their nucleotide sequences determined by methods involving complete and further partial digestion with ribonucleases A and T(1). Fragment 20 had a sequence that coded for the amino acids in positions 32-53 of the coat protein of the bacteriophage. Fragment 20X, on further purification in 7m-urea, gave rise to two smaller nucleotides whose sequences coded for the amino acids in positions 56-66 and 67-76 of the coat protein. The sequence of the two fragments was such that they could be written in the form of loops stabilized by base-pairing.
Article
A method is described for the two-dimensional fractionation of ribonuclease digests of 32P-labelled RNA. High-voltage ionophoresis is used in both dimensions. The first is on cellulose acetate at pH 3·5, the second on DEAE-paper at an acid pH. The method is capable of resolving the di- and tri- and most of the tetra-nucleotides in digests prepared by the action of ribonuclease T1 or pancreatic ribonuclease. It has been applied to the 16 s and 23 s components of ribosomal RNA which show significant quantitative differences, and to sRNA from Escherichia coli and from yeast. A method is described for the determination of the sequence of a nucleotide by partial digestion with spleen phosphodiesterase.
Rue Reni-Descartes, Esplanade. F-67000 Strdsbourg, France Eur
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