Cruciform structure of a DNA motif of parvovirus minute virus of mice (prototype strain) involved in the attenuation of gene expression (original) (raw)
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Anchoring Nascent RNA to the DNA Template Could Interfere with Transcription
Biophysical Journal, 2011
During normal transcription, the nascent RNA product is released from the DNA template. However, in some cases, the RNA remains bound or can become reattached to the template DNA duplex (for example, through R-loop formation). We have analyzed the effect on transcription elongation of nascent RNA anchoring to the template DNA duplex. Because the RNA polymerase follows a helical path along DNA duplex during transcription, the anchoring would result in wrapping the nascent RNA around the DNA in the region between the anchoring point and the translocating polymerase. This wrapping would cause an unfavorable loss of conformation entropy of the nascent RNA. It consequently would create an apparent force to unwrap the RNA by disrupting either the transcription complex or the anchoring structure. We have estimated that this force would be comparable to those required to melt nucleic acid duplexes or to arrest transcription elongation in single-molecule experiments. We predict that this force would create negative supercoiling in the DNA duplex region between the anchoring point and the transcribing RNA polymerase: this can promote the formation of unusual DNA structures and facilitate RNA invasion into the DNA duplex. Potential biological consequences of these effects are discussed.
Molecular and Cellular Biology, 1991
We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or He...
Proceedings of the National Academy of Sciences, 1981
The 5' termini of the principal early mRNAs produced in cells transformed by wild-type simian virus 40 lie 21-25 nucleotides downstream from an A-T-T-T-A-T sequence on the DNA template. The 5' termini of early mRNAs produced by five origin-defective mutants containing deletions downstream from the A-T-T-T-A-T sequence and one viable mutant dl892 with a deletion starting 15 nucleotides upstream from this sequence were determined by a method involving synthesis, separation, and determination of the sequences of DNAs complementary to 5' termini. Mutant dl892 produced early mRNAs with the same principal 5' termini as wild-type virus; the origin-defective mutants produced mRNAs with principal 5' termini shifted downstream by a distance equivalent to the length of the deleted DNA segment. These data suggest that a DNA sequence of 29 nucleotides, which includes the A-T-T-T-A-T sequence, contains a component(s) of a promoter for early transcription. This component functi...
Initial Transcription by RNA Polymerase Proceeds Through a DNA-Scrunching Mechanism
Science, 2006
Using fluorescence resonance energy transfer to monitor distances within single molecules of abortively initiating transcription initiation complexes, we show that initial transcription proceeds through a "scrunching" mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA into itself and past its active center. We show further that putative alternative mechanisms for RNAP-active-center translocation in initial transcription, involving "transient excursions" of RNAP relative to DNA or "inchworming" of RNAP relative to DNA, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of interactions between RNAP and promoter DNA and between RNAP and initiation factors during promoter escape.
Nucleic Acids Research, 2011
We present evidence that the reverse transcriptase (RT) of human immunodeficiency virus type-1 stabilizes in vitro very short (2-nt) duplexes of 3 0-overhangs of the primer strand that are annealed to complementary dinucleotides tails of DNA or RNA template strands, provided that these sequences contain at least one C or G. This RT-induced strand 'clamping' activity promotes RT-directed DNA synthesis. This function is achieved only when the functional template strand is adjacent to a second DNA or RNA segment, annealed upstream to most of the primer (without gaps). The combined clamp/polymerase activity is typical to RTs, as it was found in different RTs from diverse retroviral groups, whereas cellular DNA-polymerases (devoid of 3 0 !5 0 exonucleolytic activity) showed no clamp activity. The clampassociated DNA-binding activity is markedly stabilized by dGTP, even when dGTP is not incorporated into the nascent DNA strand. The hereby-described function can help RTs in bridging over nicks in the copied RNA or DNA templates, encountered during reverse transcription. Moreover, the template-independent blunt-end synthesis of RTs can allow strand transfers onto compatible acceptor strands while synthesizing DNA. These RT properties can shed light on potentially-new roles of RTs in the reversetranscription process and define new targets for anti-retroviral drugs. cleaves the RNA template in the generated RNA/DNA duplex (4). DNA synthesis produces both (À) and (+) DNA strands, while RNase H removes the tRNA-primer and the viral genomic RNA template (1,3-5). During RTN, there are two strand transfers, or template switches events, where the 3 0-end of the elongated DNA primer switches to a second template (1-3,5). In the first one, designated (À) strand transfer, the DNA is translocated onto the 3 0-end of the genomic RNA. In the second one, (+) strand transfer, the 3 0-end of the (+) strand with the primer-binding site (PBS) sequence switches to a complementary sequence in the (À) DNA strand. These transfers depend on stable sequence complementarities between the ends of the growing (donor) DNA and the acceptor RNA or DNA strands. These complementary sequences are relatively long. In human immunodeficiency virus type-1 (HIV-1), the terminal repeat (R), which promotes (À) strand transfer, is 98-nt long and in murine leukemia virus (MLV), it is 68-nt long, whereas the PBS, is usually 18-nt long in all retroviruses that use a tRNA primer (including HIV-1 and MLV) (1,6). We present here in vitro evidence that RTs can perform template switches even with a very short (2-nt) complementarity between the 3 0-ends of the primer donor strand and the DNA or RNA template acceptor strands. These dinucleotide duplexes are markedly stabilized by RT that 'clamps' together these otherwise unstable duplexes. This stabilization of sequence micro-homology efficiently promotes DNA synthesis. The apparently-new
Journal of Virology, 2000
The nonsegmented negative-strand RNA (NNS) viruses have a single-stranded RNA genome tightly encapsidated by the viral nucleocapsid protein. The viral polymerase transcribes the genome responding to specific gene-start and gene-end sequences to yield a series of discrete monocistronic mRNAs. These mRNAs are not produced in equimolar amounts; rather, their abundance reflects the position of the gene with respect to the single 3-proximal polymerase entry site. Promoter-proximal genes are transcribed in greater abundance than more distal genes due to a localized transcriptional attenuation at each gene junction. In recent years, the application of reverse genetics to the NNS viruses has allowed an examination of the role of the gene-start and gene-end sequences in regulating mRNA synthesis. These studies have defined specific sequences required for initiation, 5 modification, termination, and polyadenylation of the viral mRNAs. In the present report, working with Vesicular stomatitis virus, the prototypic Rhabdovirus, we demonstrate that a gene-end sequence must be positioned a minimal distance from a gene-start sequence for the polymerase to efficiently terminate transcription. Gene-end sequences were almost completely ignored in transcriptional units less than 51 nucleotides. Transcriptional units of 51 to 64 nucleotides allowed termination at the gene-end sequence, although the frequency with which polymerase failed to terminate and instead read through the gene-end sequence to generate a bicistronic transcript was enhanced compared to the observed 1 to 3% for wild-type viral mRNAs. In all instances, failure to terminate at the gene end prevented initiation at the downstream gene start site. In contrast to this size requirement, we show that the sequence between the gene-start and gene-end signals, or its potential to adopt an RNA secondary structure, had only a minor effect on the efficiency with which polymerase terminated transcription. We suggest three possible explanations for the failure of polymerase to terminate transcription in response to a gene-end sequence positioned close to a gene-start sequence which contribute to our emerging picture of the mechanism of transcriptional regulation in this group of viruses.
Journal of Virology, 2005
The generation of subgenomic mRNAs in coronavirus involves a discontinuous mechanism of transcription by which the common leader sequence, derived from the genome 5 terminus, is fused to the 5 end of the mRNA coding sequence (body). Transcription-regulating sequences (TRSs) precede each gene and include a conserved core sequence (CS) surrounded by relatively variable sequences (5 TRS and 3 TRS). Regulation of transcription in coronaviruses has been studied by reverse-genetics analysis of the sequences immediately flanking a unique CS in the Transmissible gastroenteritis virus genome (CS-S2), located inside the S gene, that does not lead to detectable amounts of the corresponding mRNA, in spite of its canonical sequence. The transcriptional inactivity of CS-S2 was genome position independent. The presence of a canonical CS was not sufficient to drive
Nucleic Acids Research, 1983
Four virus-specific transcripts have been identified in murine cells infected with Minute-Virus-of-Mice (MVM). These RNAs, 4.8, 3.3, 3.0 and 1.8 kilobases in length, designated Rl to R4 respectively, are all transscribed from the virion (-) strand of DNA and they are all polyadenylated and spliced. The Rl transcript is derived from sequences that reside on the genome between 4.0 and 95 map units (mu). Transcript R2 is composed of exon sequences derived from mu coordinates 4.0-10.0, 40-46 and 48-95. The most abundant RNA, R3, is transcribed from sequences mapping between 40 and 95 mu. All three of these RNAs have a short intron sequence between 46-48 mu removed. The least abundant transcript, R4, has not been mapped precisely, however it hybridizes with all three EcoRI fragments which span the entire 5 kb genome. In vitro transcription of cloned restriction fragments of MVM DNA confirm the existence of functional promoters at map coordinates 4.0 and 39 and sequence analysis of these regions of the viral DNA reveal the characteristic features of RNA polymerase II promoters. These results indicate that MVM DNA encodes two overlapping transcription units with separate promoters near the left end (4.0 mu) and middle (39 mu) of the genome.
The effect of template RNA structure on elongation by HIV-1 reverse transcriptase
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1999
Reverse transcription of the RNA genome of retroviruses has to proceed through some highly structured regions of the template. The RNA genome of the human immunodeficiency virus type 1 (HIV-1) contains two hairpin structures within the repeat (R) region at the 5P end of the viral RNA . These structures, the TAR and polyA hairpins, fulfil important functions in the viral life cycle. We analyzed the in vitro elongation properties of the HIV-1 reverse transcriptase (RT) enzyme on the wild-type RNA template and mutants thereof with either a stabilized or a destabilized polyA hairpin. Stable RNA structure was found to interfere with efficient elongation of the RT enzyme, as judged by the appearance of pause cDNA products. A direct relation was measured between the stability of template RNA structure and the extent of RT pausing. However, the position of structure-induced pause sites is rather diverse, with significant stops at a position approximately 6 nt ahead of the basepaired stem of the TAR and polyA hairpins. This suggests that the RT enzyme is stalled when its most forward domain contacts the RNA duplex. Addition of the viral nucleocapsid protein (NC) to the in vitro assay was found to overcome such structure-induced RT stops. These results indicate that the RT polymerase has problems penetrating regions of the template with stable RNA structure. This effect was more pronounced at high Mg 2 concentrations, which is known to stabilize RNA secondary structure. Such a structure-induced defect was not apparent in reverse transcription assays performed in virus-infected cells, which is either caused by the NC protein or other components of the virion particle. Thus, retroviruses can use relatively stable RNA structures to control different steps in the viral life cycle without interfering with the process of reverse transcription. ß 1999 Elsevier Science B.V. All rights reserved.