Showing papers on "RNA-dependent RNA polymerase published in 1981"

Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA

Abstract: Transcription of ColE1 DNA by RNA polymerase in vitro starts at two sites in a region required for maintenance of the plasmid. Certain transcripts that start at one of the sites can be cleaved by RNase H and then act as primers for DNA replication. Transcription from the other site produces a RNA approximately 108 nucleotides long (species I or RNA I). Transcripts analogous to the primer and RNA I of ColE1 are produced when p15A or small derivatives of two other ColE1-compatible plasmids, CloDF13 and RSF1030, are used as template. If purified RNA I is added to the transcription reaction containing RNase H, formation of primer is inhibited. Each RNA I can inhibit primer formation by the plasmid that specifies it but has no effect on primer formation by heterologous templates. Thus, the inhibition of primer formation by RNA I is incompatibility specific. Because RNA I does not inhibit initiation or propagation of transcription or the processing of preformed precursors, the step that is sensitive to inhibition is probably formation of the hybrid between the primer precursor and the template. This hybrid is the required substrate for RNase H. Experiments with recombinant plasmids show the region that determines the specificity of response to RNA I to be greater than 300 base pairs upstream of the origin of DNA replication.

Plasmid ColE1 incompatibility determined by interaction of RNA I with primer transcript

Abstract: Mutants of plasmid pNT7 that can coexist with plasmid pMB9 in growing bacteria have been isolated. These mutants show altered incompatibility properties and increased copy numbers. Each mutant has a single base change at or near the center of one of the three palindromes in the region that specifies two RNA species: a larger one (primer transcripts) that provides a primer for DNA replication and a smaller one (RNA I) that is the incompatibility-specific inhibitor of primer formation. In vitro transcription studies show that the single base changes affect both the ability of RNA I to inhibit primer formation and the sensitivity of primer formation to inhibition by RNA I. RNA I hybridizes to the primer transcript, and the rate of hybridization is reduced by the single base changes. Based on analyses of inhibition of in vitro primer formation by RNA I and of in vivo properties of the mutant plasmids, we conclude that incompatibility between two plasmids can be attributed to inhibition of primer formation on one of the plasmids by the RNA I of the other. Inhibition of primer formation by RNA I appears to be the mechanism that determines the copy number of pNT7 and its derivatives.

Polyadenylation sites for influenza virus mRNA.

Abstract: Polyadenylated transcripts of influenza virus RNA are incomplete copies of the individual genome segments, lacking sequences complementary to the 5'-terminal nucleotides of the virion RNA. By using a procedure which depends on the polyadenylic acid tail of the mRNA being encoded in part by the genome, we have determined that the common tract of uridine residues, approximately 17 to 22 nucleotides from the 5' end of each segment, is the site of polyadenylation of influenza virus mRNA.

RNA is synthesized at the nuclear cage

Abstract: The photomicrographs of ‘genes in action’ taken by Miller and colleagues strikingly illustrate transcription of eukaryotic genes by RNA polymerase moving along the DNA1,2. Although completed transcripts may be associated with sub-nuclear structures3–7, the view that a mobile polymerase processes along the DNA remains unchallenged. Here we examine an alternative view—that transcription occurs as DNA passes through a transcription complex fixed to a sub-nuclear structure, and we show that transcribed sequences are closely associated with a sub-nuclear structure that we call a nuclear cage.

A small nuclear ribonucleoprotein is required for splicing of adenoviral early RNA sequences.

Abstract: The size and structure of viral RNA species synthesized in nuclei isolated during the early phase of productive infection by adenovirus type 2 have been examined by electrophoresis in denaturing polyacrylamide cells and the nuclease S1 assay. The major products of transcription in vitro of early regions 1 and 2 in the adenoviral genome are processed RNA molecules that appear to be correctly spliced in isolated nuclei. Splicing of adenoviral RNA molecules is inhibited when nuclei are preincubated with antibodies from systemic lupus erythematosus patients that immunoprecipitate small nuclear ribonucleoprotein particles. The specificity of these antibodies suggests that ribonucleoprotein particles containing U1 RNA are required for splicing of the adenoviral RNA sequences we have examined.

Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable.

Abstract: The synthesis rates and half-lives of the individual mitochondrial ribosomal ribonucleic acid (RNA) and polyadenylic acid-containing RNA species in HeLa cells have been determined by analyzing their kinetics of labeling with [5-^3H]-uridine and the changes in specific activity of the mitochondrial nucleotide precursor pools. In one experiment, a novel method for determining the nucleotide precursor pool specific activities, using nascent RNA chains, has been utilized. All mitochondrial RNA species analyzed were found to be metabolically unstable, with half-lives of 2.5 to 3.5 h for the two ribosomal RNA components and between 25 and 90 min for the various putative messenger RNAs. A cordycepin "chase" experiment yielded half-life values for the messenger RNA species which were, in general, larger by a factor of 1.5 to 2.5 than those estimated in the labeling kinetics experiments. On the basis of previous observations, a model is proposed whereby the rate of mitochondrial RNA decay is under feedback control by some mechanism linked to RNA synthesis or processing. A short half-life was determined for five large polyadenylated RNAs, which are probably precursors of mature species. A rate of synthesis of one to two molecules per minute per cell was estimated for the various H-strand-coded messenger RNA species, and a rate of synthesis 50 to 100 times higher was estimated for the ribosomal RNA species. These data indicate that the major portion of the H-strand in each mitochondrial deoxyribonucleic acid molecule is transcribed very infrequently, possibly as rarely as once or twice per cell generation. Furthermore, these results are consistent with a previously proposed model of H-strand transcription in the form of a single polycistronic molecule.

Mouse hepatitis virus A59: mRNA structure and genetic localization of the sequence divergence from hepatotropic strain MHV-3.

Abstract: The composition and structure of the mouse hepatitis virus (MHV)-specific RNA in actinomycin D-treated, infected L-2 cells were studied. SEven virus-specific RNA species with molecular weights of 0.6 X 10(6), 0.9 X 10(6), 1.2 X 10(6), 1.5 X 10(6), 3.0 X 10(6), 4.0 X 10(6), and 5.4 X 10(6) (equivalent to the viral genome) were detected. T1 oligonucleotide fingerprinting studies suggested that the sequences of each RNA species were totally included within the next large RNa species. The oligonucleotides of each RNA species were mapped on the 60S RNA genome of the virus. Each RNA species contained the oligonucleotides starting from the 3' end of the genome and extending continuously for various lengths in the 3' leads to 5' direction. All of the viral RNA species contained a polyadenylate stretch of 100 to 130 nucleotides and probably identical sequences immediately next to the polyadenylate. These data suggested that the virus-specific RNAs are mRNA's and have a stairlike structure similar to that of infectious bronchitis virus, an avian coronavirus. A proposal is presented, based on the mRNA structure, for the designation of the genes on the MHV genome. Using this proposal, the sequence differences between A59, a weakly pathogenic strain, and MHV-3, a strongly hepatotropic strain, were localized primarily in mRNA's 1 and 3, corresponding t genes A and C.

Clustering of RNA polymerase B molecules in the 5' moiety of the adult beta-globin gene of hen erythrocytes.

Abstract: Nuclei were prepared from mature and immature hen erythrocytes and incubated for RNA synthesis in the absence or in the presence of Sarkosyl. The in vitro labelled synthesized RNA was hybridized to specific 5' and 3' fragments of the chicken adult beta-globin gene to investigate the possible presence of RNA polymerase molecules bound to this gene in the form of transcriptional complexes. Surprisingly, such RNA polymerase B molecules were found located preferentially in the 5' end moiety of the beta-globin genes of mature erythrocytes, although they are apparently evenly distributed along the beta-globin genes of immature polychromatic erythrocytes. The significance of these observations with respect to (1) preferential DNaseI sensitivity of "genes which have been transcribed" and (2) control of transcription in eukaryotic cells is discussed.

Comparative analysis of RNA genomes of mouse hepatitis viruses.

Abstract: The RNA genomes of various murine hepatitis virus (MHV) strains were studied by T1-oligonucleotide fingerprinting analysis with regard to their structure and sequence relationship. It was found that the MHV particles contained only positive-stranded 60S RNA which had a "cap" structure at its 5' end. No negative-stranded RNA was found. It was also shown that most of the MHV strains had diverged quite extensively in their genetic sequences. However, MHV-3, a hepatotropic strain, and A59, a nonpathogenic strain, were found to have very similar oligonucleotide fingerprinting patterns. Yet, each of them contained two to four specific oligonucleotides. The MHV-3-specific oligonucleotides were conserved in almost all of the hepatotropic MHV strains studied. In contrast, two of the A59-specific oligonucleotides were absent from the genomes of all hepatotropic strains. These findings suggest that these unique oligonucleotides might be localized at the genetic region(s) associated with viral pathogenicity or other biological properties of the virus. Comparison of viral structural proteins also suggests that MHV-3 and A59 are more closely related than other MHV strains. The significance of these findings is discussed.

Wild-type tRNATyrG reads the TMV RNA stop codon, but Q base-modified tRNATyrQ does not.

Abstract: Although protein synthesis usually terminates when a stop codon is reached along the messenger RNA sequence, there are examples, mainly in viruses, of the stop codon being suppressed by a tRNA species. A strong candidate for this phenomenon occurs in tobacco mosaic virus (TMV) in the form of two proteins (110K and 160K, of molecular weights 110,000 and 160,000, respectively)1, sharing an N-terminus sequence, which are translated in vitro from a purified species of viral RNA. We have investigated the identity of the tRNA responsible for production of the 160K protein and show here that it is one of the tyrosine tRNAs. Another tyrosine tRNA, in which the first base of the anticodon is highly modified, does not act as a suppressor, indicating the possible regulatory function of such modifications.

DNA-dependent RNA polymerase II of plant origin transcribes viroid RNA into full-length copies.

Abstract: DNA-dependent RNA polymerase II purified from healthy plant tissue is capable of synthesizing linear (-)-viroid RNA copies of full length from (+)-viroid RNA templates in vitro. Together with the specific alpha-amanitin sensitivity of viroid replication observed in vivo, these findings suggest that viroids replicate by an entirely novel mechanism in which infecting viroid RNA molecules are copied by the host enzyme which is normally responsible for the synthesis of nuclear precursors to messenger RNA.

Characterisation of cauliflower mosaic virus DNA sequences which encode major polyadenylated transcripts

Abstract: Cauliflower mosaic virus DNA sequences which encode two major polyadenylated RNA species, the 1.9 kb messenger RNA for the 62,000 MW virus inclusion body polypeptide and 35S RNA, were mapped using the nuclease 51 procedure. The 1.9 kb RNA has an eleven nucleotide leader sequence transcribed from alpha-strand DNA located at co-ordinate 0.72 m.u. (map units), immediately upstream of the AUG initiation codon of reading frame VI. The 3'-end of 1.9 kb RNA maps at 0.95 m.u. and is co-terminal with the 3'-end of 35S RNA, a complete transcript of the DNA alpha-strand. The 5'-end of 35S RNA maps at 0.93 m.u. and is located upstream of the discontinuity G1 (zero m.u.) in the alpha-strand and some 200 nucleotides upstream of the sequence encoding its own 3'-terminus.

Transcriptional control regions: nucleotide sequence requirements for initiation by RNA polymerase II and III.

Abstract: Until recently, the nucleotide sequences involved in the initiation of transcription within eukaryotic cells remained a mystery. Now, thanks to a variety of technological advances, it is possible to identify these sequences and appreciate their functions in the transcription process. Cloning technology enables one to isolate and study genes individually. Rapid DNA and RNA sequencing procedures facilitate nucleotide sequence analysis of both the genes and the RNAs they encode. A variety of in vitro mutagenesis protocols makes it possible to direct deletions and single base-pair changes to specific nucleotide sequences. Selection procedures are available to return genes which have been manipulated in vitro to cells for analysis. And, finally, in vitro systems have been developed which accurately transcribe either RNA polymerase II- or polymerase Ill-type genes. Genes can now be isolated, sequenced, mutated, and their transcription studied both in vivo and in vitro. As a result, specific functions in the transcription process can be related to specific nucleotide sequences.

Control region for adenovirus VA RNA transcription

Abstract: Plasmids containing the VA RNA genes of adenovirus are faithfully transcribed by a crude cytoplasmic extract containing DNA-dependent RNA polymerase III [Wu, G.-J. (1978) Proc. Natl. Acad. Sci. USA 75, 2175-2179]. By subjecting these DNA templates to in vitro site-directed mutagenesis with a novel enzyme of Pseudomonas and recloning in pBR322, we have constructed an ordered series of deletions which affect the in vitro transcription of the major RNA polymerase III viral product, VAI RNA. Three regions that are required for specific synthesis of VAI RNA can be defined. One, inside the gene at nucleotides +10 to +76, affects the transcription in an all-or-none fashion. Transcription is initiated on plasmid sequences that replace up to 10 nucleotides downstream from the 5' end of the gene. Variants with deletions past nucleotide +15 do not support the transcription of VAI RNA. Removal of 3'-end sequences downstream from +76 allows correct initiation. A second region, upstream from the initiation site, affects the exact alignment of the first nucleotide of the transcript [Thimmapaya, B., Jones, N. & Shenk, T. (1979) Cell 18, 947-959]. A third region, downstream from +76, encodes signals for termination of transcription, and new signals were brought in with other viral DNA sequences. Transcription competition experiments indicate that the primary site for binding of a transcriptional regulation factor is located between nucleotides +55 and +70 and suggest that the control region is bifunctional. An internal control region for VAI RNA, approximately 60 bases long and 11 bases downstream from the 5' end of the gene, can be defined.

Pausing of RNA polymerase during in vitro transcription of the tryptophan operon leader region.

Abstract: RNA polymerase molecules pause at a single site during in vitro transcription of the tryptophan (trp) operon leader region. Pausing was observed when DNA templates derived from Escherichia coli. Salmonella typhimurium, and Klebsiella aerogenes were used. Fingerprint analyses showed that the major RNA species produced by the transcriptional pause is 91 nucleotides long. A minor RNA species 90 nucleotides long was also detected. Single-round transcription experiments were used to study the kinetics of pausing. Time course, pulse-chase, and delayed-labeling experiments suggest that every RNA polymerase molecule transcribing the trp leader region pauses. A suboptimal ribonucleoside triphosphate concentrations, the half-life of paused-leader RNA was approximately 3 min at 22 degrees C and 0.7 min at 37 degrees C. At near-optimal ribonucleoside triphosphate concentrations, the half-time of the paused species dropped to about 0.3 min at 22 degrees C. The appearance and half-life of the paused species were unaffected by salt concentration, rho factor, guanosine 3'-5'-bis(diphosphate), or point mutations in the trp attenuator region. It is postulated that transcriptional pausing may play a role in maintaining the synchronization of transcription and translation that is vital in the control of transcription termination at the trp operon attenuator.

Poliovirus replication proteins: RNA sequence encoding P3-1b and the sites of proteolytic processing

Abstract: A partial amino-terminal amino acid sequence of each of the major proteins encoded by the replicase region (P3) of the poliovirus genome has been determined. A comparison of this sequence information with the amino acid sequence predicted from the RNA sequence that has been determined for the 3' region of the poliovirus genome has allowed us to locate precisely the proteolytic cleavage sites at which the initial polyprotein is processed to create the poliovirus products P3-1b (NCVP1b), P3-2 (NCVP2), P3-4b (NCVP4b), and P3-7c (NCVP7c). For each of these products, as well as for the small genome-linked protein VPg, proteolytic cleavage occurs between a glutamine and a glycine residue to create the amino terminus of each protein. This result suggests that a single proteinase may be responsible for all of these cleavages. The sequence data also allow the precise positioning of the genome-linked protein VPg within the precursor P3-1b just proximal to the amino terminus of polypeptide P3-2.