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At least 1,045 records · Page 58Linked to original sources
PubMed2001

Identification of a strong promoter of bacteriophage MB78 that interacts with a host coded factor and regulates the expression of a structural protein.

A strong promoter of bacteriophage MB78 which controls the expression of a small structural protein of the phage has been identified and characterized. Analysis of its nucloetide sequence upstream of the translational start site revealed the presence of conserved -10 (TAATAT) and -35 (TTCTCCT) regions. It was observed that transcription initiates with thymidine residue, 86 nt upstream of the translational start site. Transcription seems to undergo alternate up and down regulation. This promoter is efficiently recognized by sigma 70 RNA polymerase and a host factor also binds to it. Binding of RNA polymerase is independent of binding of the host factor.

Amino Acid Sequence↗
PubMed1994

Context-dependent effects of upstream A-tracts. Stimulation or inhibition of Escherichia coli promoter function.

Phased A-tract sequences were inserted in the upstream region of three synthetic promoters known to encompass different rate-limiting steps within the pathway of RNA polymerase-promoter interaction (Ellinger et al., accompanying paper). Promoter PS1, which is rate-limited in complex formation, was stimulated by A-tracts in vivo. Permanganate probing showed that the stimulation is due to an enhanced ability to compete for limiting RNA polymerase in vivo, leading to the increased formation of open complexes. By contrast, promoters PS2 and PS3, which are rate-limited in steps following open complex formation, were inhibited in vivo by A-tracts. Permanganate probing showed that the inhibition was accompanied by an A-tract-dependent accumulation of stalled initial transcribing complexes. A single A-tract was as effective as three. The phasing of the A-tracts with respect to the core promoter sequence was found to be important for promoter function. The position that caused maximal activation at one promoter caused maximal inhibition at another. These results suggest that the same molecular interaction gives rise to both inhibition and activation. This is likely to be due to facilitated RNA polymerase binding in the presence of A-tracts, which stimulates binding-limited promoters but inhibits promoter function in which polymerase escape and promoter clearance is rate limiting.

Base Sequence↗
PubMed1995

Interference of PR-bound RNA polymerase with open complex formation at PRM is relieved by a 10-base pair deletion between the two promoters.

Bacteriophage lambda promoters PR and PRM direct RNA synthesis in divergent orientations from start sites 82 base pairs apart. We had previously determined that the presence on the same DNA fragment of a wild type PR promoter interfered with the utilization of the PRM promoter. The results reported here concern the effects of changing the distance between the start sites by insertion or deletion of 5 or 10 base pairs. Three different techniques (run-off transcription, gel mobility shift, and permanganate probing) were employed to monitor complex formation at PRM. Unexpectedly we find that deletion of 10 base pairs between the start sites abolishes the interference, whereas insertion of 10 base pairs does not. Deletion of 5 base pairs, however, essentially prevents joint complex formation at PR and PRM. These findings suggest several ways in which for the wild type separation of the two promoters the utilization of PRM could be affected by an RNA polymerase at PR. In addition to direct steric interference, these include the obstruction of access to DNA sites necessary for optimal contact with the RNA polymerase.

Bacteriophage lambda↗
PubMed1984

Direct repeats surrounding the ribosomal RNA genes of Physarum polycephalum.

Sequence homology was found between the external transcribed spacer and the terminal non-transcribed spacer of Physarum polycephalum rDNA. The homologous sequences were located 2kb upstream from the 19s rRNA gene and 0.3kb downstream from 26S rRNA gene, respectively, and were arranged in a direct repeat manner. Sequence analyses showed that the direct repeats consisted of two parts: one was sequences of about 130bp which showed over 90% sequence homology with each other. The other consisted mainly of many tandem repeats of a 50 to 52bp unit. The direct repeat-rRNA genes-direct repeat unit was found to be flanked by short direct repetitious sequences. Based on these findings, the significance of the direct repeat is discussed in terms of evolution of rDNA.

Base Composition↗
PubMed1993

An 'elaborated' pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA.

The RNA polymerase gene (gene 1) of the human coronavirus 229E is approximately 20 kb in length and is located at the 5' end of the positive-strand genomic RNA. The coding sequence of gene 1 is divided into two large open reading frames, ORF1a and ORF1b, that overlap by 43 nucleotides. In the region of the ORF1a/ORF1b overlap, the genomic RNA displays two elements that are known to mediate (-1) ribosomal frameshifting. These are the slippery sequence, UUUAAAC, and a 3' pseudoknot structure. By introducing site-specific mutations into synthetic mRNAs, we have analysed the predicted structure of the HCV 229E pseudoknot and shown that besides the well-known stem structures, S1 and S2, a third stem structure, S3, is required for a high frequency of frameshifting. The requirement for an S3 stem is independent of the length of loop 2.

Amino Acid Sequence↗
PubMed1992

Specific inhibition of transcription by triple helix-forming oligonucleotides.

Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation. The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site. A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro. We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature. The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix. Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition. Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action. The 13-mer oligonucleotide containing a psoralen derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm.

Base Sequence↗
PubMed1993

Expression of human alpha 1 interferon genes in vectors containing tandemly located promoters recognized by two different RNA polymerases (Escherichia coli and T7).

An expression vector containing two tandemly located promoters (T7 and P1) and two transcription terminators recognized by two different RNA polymerases (T7 RNA polymerase and Escherichia coli RNA polymerase) was constructed. Human alpha 1 interferon gene variants were cloned in this vector and their expression was studied in E. coli strains containing [E. coli BL2I (DE3)] or devoid (E. coli BL21) of the gene for the T7 RNA polymerase. We report that simultaneous activity of the two promoters reduces the level of gene expression when compared with the levels of expression corresponding to either P1 or T7 promoter alone.

Base Sequence↗
PubMed1990

Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter.

A G----T mutation at the start-point of transcription of the phage P22 sar promoter (sar + 1T) causes a novel defect in promoter clearance by Escherichia coli RNA polymerase (RNAP) in vitro. Under standard transcription conditions, in the presence of high concentrations of all four NTPs, the predominant products from this promoter are poly(U) chains of varying length. Because the mutation creates a run of four T: A base-pairs from - 1 to +3 (TGTT----TTTT), we propose that synthesis of poly(U) is pseudo-templated by the A4 stretch on the template strand. G----A and G----C mutations at position +1 do not cause pseudo-templated transcription. Several molecules of poly(U) are produced and released per sar+1T promoter-polymerase complex without dissociation of RNAP from the template DNA. The exponential relationship between yield and size of individual poly(U) species indicates that there is a constant probability that another U residue will be added to the nascent chain. Presumably, pseudo-templated transcription occurs by a slippage (stuttering) mechanism like that proposed to explain certain kinds of RNA editing in eukaryotic viral mRNAs.

Base Sequence↗
PubMed2006

Escherichia coli RNA polymerase recognition of a sigma70-dependent promoter requiring a -35 DNA element and an extended -10 TGn motif.

Escherichia coli sigma70-dependent promoters have typically been characterized as either -10/-35 promoters, which have good matches to both the canonical -10 and the -35 sequences or as extended -10 promoters (TGn/-10 promoters), which have the TGn motif and an excellent match to the -10 consensus sequence. We report here an investigation of a promoter, P(minor), that has a nearly perfect match to the -35 sequence and has the TGn motif. However, P(minor) contains an extremely poor sigma70 -10 element. We demonstrate that P(minor) is active both in vivo and in vitro and that mutations in either the -35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the -10 element more canonical, thus converting the -35/TGn promoter to a -35/-10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with P(minor), the DNA is single stranded from positions -11 to +4. We also demonstrate that transcription from P(minor) incorporates nontemplated ribonucleoside triphosphates at the 5' end of the P(minor) transcript, which results in an anomalous assignment for the start site when primer extension analysis is used. P(minor) represents one of the few -35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized -10/-35 and extended -10 promoters used by E. coli RNA polymerase.

Base Sequence↗
PubMed1993

Peptide nucleic acids (PNAs): potential antisense and anti-gene agents.

The binding of peptide nucleic acids (PNAs) T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 to double-stranded DNA targets A10, A5GA4 and A2GA2GA4 was studied by nuclease S1 probing. It is found that the PNAs bind preferentially to their complementary targets, weaker to targets containing one mismatch and not to targets containing two mismatches. Using an RNA polymerase T3 in vitro transcription system, it is found that a PNA T10-LysNH2 bound downstream from the promoter causes transcription elongation arrest at the PNA binding site only when the PNA is bound to the template strand. Finally, it is shown that primer extension by Taq DNA polymerase on a single-stranded template is arrested at an occupied PNA T10 binding site. These results are discussed in relation to PNAs as potential anti-sense and anti-gene drugs.

Base Sequence↗
PubMed1991

Antitermination of vaccinia virus early transcription: possible role of RNA secondary structure.

Transcription of vaccinia early genes by the viral RNA polymerase terminates downstream of a signal sequence TTTTTNT in the nontemplate DNA strand. Signal recognition occurs at the level of the sequence UUUUUNU in nascent RNA and depends on a virus-encoded termination factor (VTF). The presence of TTTTTNT elements within protein encoding regions of some early genes requires that these 5' proximal signals be ignored in order to achieve early expression of the full-sized proteins. In the case of the A18R gene, which contains a proximal terminator that is not utilized in vivo (Pacha et al., J. Virol. 64, 3853-3863 (1990)), the TTTTTNT sequence can be folded into a potential hairpin structure such that UUUUUNU would be part of a duplex stem in the nascent RNA. We find that the A18R putative hairpin is unable to promote factor-dependent termination in a purified in vitro transcription system. Sequence manipulations that abrogate the potential to form an RNA hairpin restore the activity of the TTTTTNT motif. The in vitro studies suggest that antitermination at the proximal site of the A18R gene may be mediated by secondary structure in the nascent RNA, and that early termination involves recognition by VTF and/or RNA polymerase of the UUUUUNU sequence in single-stranded form.

Base Sequence↗
PubMed1994

Rapid characterization of new pestivirus strains by direct sequencing of PCR-amplified cDNA from the 5' noncoding region.

Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the rapid laboratory diagnosis of pestivirus infections. A direct DNA sequencing method was developed for the analysis of the amplified cDNA from the 5' noncoding region of the viral genome. 70 pestivirus strains were compared in this study. Sequence analysis allowed the characterization of each isolate as either classical swine fever virus (CSFV), bovine viral diarrhea virus, or border disease virus, respectively. The 48 CSFV strains could be further classified into several subgroups, which correlated either with the geographical origin or the date of the first isolation of the respective isolate.

Animals↗
PubMed2003

Modification of the 5' terminus of Sindbis virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids at the N terminus to function in RNA replication.

We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40 degrees C upon addition of AU, AUA, or AUU to the 5' terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3'-UA-5' is required at the 3' terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5' end of the genomic RNA provides unpaired 3'-UA-5' at the 3' end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3'-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3' terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

5' Untranslated Regions↗
PubMed1996

Specific expression of the chloroplast gene for RNA polymerase (rpoB) at an early stage of leaf development in rice.

The rpoB gene for the beta subunit of rice chloroplast RNA polymerase was found to be highly expressed in unexpanded immature leaves that contained proplastids, indicating the specific expression of rpoB at an early stage of chloroplast development. A putative transcription start site (tss) was identified, but the 5' upstream region of the tss had no sequences resembling typical --35 and --10 elements. A palindromic sequence and high AT-content were recognized.

Base Sequence↗
PubMed1995

Polymerase activity of in vitro mutated rabies virus L protein.

The large (L) protein of nonsegmented negative-strand RNA viruses is the multifunctional catalytic component of the viral ribonucleoprotein (RNP) complex. To address the role of conserved rabies virus (RV) L protein sequences predicted to be involved in RNA polymerase activity, a reverse genetics approach was applied that allows intracellular reconstitution of transcriptionally active RV RNPs from plasmid-encoded proteins. Artificial RV model genomes encoding bacterial chloramphenicol acetyltransferase or firefly luciferase was used to determine the polymerase activity of a series of 23 RV L proteins mutated in the highly conserved C motif of the proposed polymerase module. All constructs with mutations of the GDN core sequence of motif C, which is proposed to be a variant of the catalytical XDD residues of RNA polymerase and reverse transcriptases, failed to express the reporter genes. In addition, the identity of the upstream residues AQ was crucial for maintenance of polymerase activity. Several conservative and nonconservative mutations introduced into the three amino acids QVL located downstream of the GDN core resulted in reduced polymerase activities and expression of luciferase in the range 0.4 to 92% compared to the parental L protein.

Amino Acid Sequence↗
PubMed2001

Translocation of sigma(70) with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA.

Using fluorescence resonance energy transfer, we show that, in the majority of transcription complexes, sigma(70) is not released from RNA polymerase upon transition from initiation to elongation, but, instead, remains associated with RNA polymerase and translocates with RNA polymerase. The results argue against the presumption that there are necessary subunit-composition differences, and corresponding necessary mechanistic differences, in initiation and elongation. The methods of this report should be generalizable to monitor movement of any molecule relative to any nucleic acid.

Base Sequence↗