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At least 739 records · Page 41Linked to original sources
PubMed1999

Myelin-associated oligodendrocytic basic protein mRNAs reside at different subcellular locations.

The mRNAs for two myelin proteins, myelin basic protein (MBP) and myelin-associated oligodendrocytic basic protein (MOBP)-81A, are uniquely located at sites where myelin sheaths are assembled. Here, we use subcellular fractionation to show that four MOBP mRNAs, like MBP mRNA, are located at sites of myelin sheath assembly, and that three other MOBP mRNAs are located in oligodendrocyte soma. The MOBP-81 protein is found in myelin and in another subcellular fraction, whereas other myelin proteins, including MBP, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin-associated glycoprotein, are largely restricted to myelin. Different MBP mRNAs are generated by alternative splicing. All of them contain an RNA transport sequence (RTS) that directs them to sites in oligodendrocytes, where myelin sheaths are assembled. Consequently, all are enriched in myelin. After fractionation, four MOBP mRNAs, MOBP-71, MOBP-81A, MOBP-99, and MOBP-169 (identified in this study), are enriched in myelin. These mRNAs contain a common exon, exon 8b, which has a nucleotide sequence that is similar to MBP mRNA RTS. This sequence likely directs these mRNAs to sites of myelin sheath assembly. Three other MOBP mRNAs, MOBP-69, MOBP-81B, and MOBP-170, lack this exon. Their subcellular distribution indicates that they are largely retained in oligodendrocyte soma. We conclude that the distribution of MOBPs in oligodendrocytes is strongly influenced by alternative splicing of the corresponding mRNAs.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1991

Mature mRNA 3' end formation stimulates RNA export from the nucleus.

We have analysed nucleocytoplasmic export of mRNAs in primate cells with the aim of identifying signals promoting RNA transport. Our results demonstrate that sequences directing either histone mRNA 3' processing or cleavage/polyadenylation of mRNA stimulate nucleocytoplasmic RNA transport. To elucidate the nature of this stimulation, we engineered test gene transcripts which could obtain a mature histone 3' end by the RNA cleaving activity of a cis-acting ribozyme, thus circumventing the cellular 3' end processing machinery. However, such transcripts were found to be transport deficient and accumulated in the nuclear compartment. Our experiments provide genetic evidence that there is a linkage between 3' end formation and the export of RNA transcripts from the nucleus. Analysis of a similar series of histone mRNAs in which the mature 3' end was generated by means of ribozyme cleavage led to the discovery of a second export mechanism which relies on features specific for mature histone RNA and which can be uncoupled from the cellular processing machinery. The presence of histone mRNA sequences and of the highly conserved histone hairpin structure, positioned close to the 3' terminus, are critical determinants for this export mechanism.

Animals↗
PubMedDate not supplied

[Sequence-determined conformational changes in the coding region of the promoter DNA on transcription complex formation].

Chemical footprinting was used to study the spatial structure of bacteriophage T7 promoter D upon formation of the transcriptionally active complex with Escherichia coli RNA polymerase. Enzyme binding was shown to induce conformational changes in sites located at positions 43 and 57, several helix turns away from the transcription start. This was the first finding of a structural deformation induced by assembly of the transcription complex. The deformation was associated with specific features of the promoter nucleotide sequence, and suggested high cooperativity in the organization of the transcription complex and substantial energy perturbations caused by the enzyme.

Bacteriophage T7↗
PubMed2001

Transcript cleavage by Thermus thermophilus RNA polymerase. Effects of GreA and anti-GreA factors.

All known multisubunit RNA polymerases possess the ability to endonucleolytically degrade the nascent RNA transcript. To gain further insight into the conformational changes that govern transcript cleavage, we have examined the effects of certain anions on the intrinsic transcript cleavage activity of Thermus thermophilus RNA polymerase. Our results indicate that the conformational transitions involved in transcript cleavage, and therefore backtracking, are anion-dependent. In addition to characterizing the intrinsic cleavage activity of T. thermophilus RNA polymerase, we have identified, cloned, and expressed a homolog of the prokaryotic transcript cleavage factor GreA from the extreme thermophiles, T. thermophilus and Thermus aquaticus. The thermostable GreA factors contact the 3'-end of RNA, stimulate the intrinsic cleavage activity of T. thermophilus RNA polymerase, and increase the k(app) of the cleavage reaction 25-fold. In addition, we have identified a novel transcription factor in T. thermophilus and T. aquaticus that shares a high degree of sequence similarity with GreA, but has several residues that are not conserved with the N-terminal "basic patch" region of GreA. This protein, Gfh1, functions as an anti-GreA factor in vitro by reducing intrinsic cleavage and competing with GreA for a binding site on the polymerase.

Amino Acid Sequence↗
PubMed1989

Early transcribed sequences affect termination efficiency of Escherichia coli RNA polymerase.

We have constructed novel transcription templates in which we have fused the late gene promoters of Escherichia coli phages lambda and 82 upstream from three different rho-independent transcription terminators. Using an in vitro transcription assay and an in vivo galactokinase expression assay, we find that the initial portion of the transcribed region significantly affects the efficiency of some downstream terminators. We have identified, by deletion, substitution and point mutation analysis, sequences responsible for these increased levels of factor-independent readthrough. Since these important sequences occur within about 30 nucleotides of the RNA start site, we suggest that the initial portion of the transcript can affect termination efficiency.

Bacteriophages↗
PubMed1991

Use of in vitro protein synthesis from polymerase chain reaction-generated templates to study interaction of Escherichia coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies.

The interaction of two Escherichia coli transcription factors (sigma 54 and sigma 32) with the core RNA polymerase was studied here. We examined the core binding ability of various fragments of these two transcription factors using a novel method for the in vitro synthesis of truncated proteins in a coupled transcription-translation (S-30) system. The method uses DNA templates generated by the polymerase chain reaction to direct synthesis of precisely truncated fragments of the encoded protein. Primers for the polymerase chain reaction contain transcription and translation signals so that the resulting product can be incubated in the S-30 directly. The synthesized proteins were used for mapping of both functional domains and epitopes of monoclonal antibodies to sigma 54 and sigma 32.

Antibodies, Monoclonal↗
PubMed2000

Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro.

Tomato ringspot nepovirus RNA-1-encoded polyprotein (P1) contains the domains for the putative NTP-binding protein, VPg, 3C-like protease and a putative RNA-dependent RNA polymerase in its C-terminal region. The N-terminal region of P1, with a coding capacity for a protein (or a precursor) of 67 kDa, has not been characterized. Using partial cDNA clones, it is shown that the 3C-like protease can process the N-terminal region of P1 at a novel cleavage site in vitro, allowing the release of two proteins, X1 (located at the N terminus of P1) and X2 (located immediately upstream of the NTB domain). P1 precursors in which the protease was inactive or absent were not cleaved by exogenously added protease, suggesting that P1 processing was predominantly in cis. Results from site-directed mutagenesis of putative cleavage sites suggest that dipeptides Q(423)/G and Q(620)/G are the X1-X2 and X2-NTB cleavage sites, respectively. The putative X1 protein contains a previously identified alanine-rich sequence which is present in nepoviruses but not in the related comoviruses. The putative X2 protein contains a region with similarity to the comovirus 32 kDa protease co-factor (the only mature protein released from the N terminus of comovirus P1 polyproteins) and to the corresponding region of other nepovirus P1 polyproteins. These results raise the possibility that the presence of two distinct protein domains in the N-terminal part of the P1 polyprotein may be a common feature of nepoviruses.

Base Sequence↗
PubMed2002

Effect of A(n) tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

In the open transcription complex (RPo), Escherichia coli RNA polymerase sigma(70) and alpha subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of A(n) DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i). A(5 )within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii). A(5)(Pg) or A(8)(Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant k(a) of RPo formation is: (i.by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K(1), of the initial closed complex; and (ii). several-fold smaller at Pg and Pm owing to a strongly decreased value of K(1). For Pm, the latter parameter was found to be dependent exponentially on four Mg(2+) ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A(8).T(8) fragment of B -DNA forms a smaller number of ionic contacts with the alpha subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase alpha and sigma(70) subunits with the proximal UP subsite and the -35 region of promoters.

Adenine↗
PubMed1990

On the use of T7 RNA polymerase transcripts for physical investigation.

A few years ago we made some observations which raised questions about the accuracy with which T7 RNA polymerase transcribes templates in vitro, and the suitability of its in vitro products for biophysical study (1). The experiments described below demonstrate that there is no reason for concern; the products of T7 RNA polymerase transcription in vitro are as suitable for biophysical characterization as RNAs synthesized in vivo. It is likely that aggregation involving the transcribed portions of the T7 RNA polymerase promoter caused our initial observations.

Base Sequence↗
PubMed1991

Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 responds to combined nitrogen deprivation by forming specialized nitrogen-fixing cells at regular intervals along the filament. Genetic and biochemical studies have indicated that regulation of gene expression during differentiation occurs at the transcriptional level. As part of a characterization of RNA polymerase during differentiation, the gene encoding the 52-kDa principal sigma factor of the Anabaena sp. strain PCC 7120 vegetative-cell RNA polymerase was isolated by using an oligonucleotide probe based on the sequence of the N-terminal seven amino acids of the purified protein. sigA codes for a 390-amino-acid polypeptide that has a predicted molecular weight of 45,641. The amino acid sequence of the polypeptide encoded by sigA contains four regions corresponding to conserved domains of the principal RNA polymerase sigma factors of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). Thus, although the subunit composition of cyanobacterial RNA polymerase core differs from that of other eubacteria (G. J. Schneider and R. Haselkorn, J. Bacteriol. 170:4136-4140, 1988), the principal sigma factor of at least one cyanobacterium is typically eubacterial. In contrast to sigma 70 and sigma 43 operon organization, sigA is monocistronic and encodes two transcripts of 1.7 and 2.2 kb. The abundance of the 1.7-kb transcript remains constant under both nitrogen-replete and nitrogen-limiting conditions, whereas the 2.2-kb transcript is induced following the removal of combined nitrogen. Continued or enhanced transcription of sigA under nitrogen starvation conditions is consistent with the observation that the principal RNA polymerase in differentiating cells contains SigA.

Amino Acid Sequence↗
PubMed1981

Sequence of U1 RNA from Drosophila melanogaster: implications for U1 secondary structure and possible involvement in splicing.

U1 RNA from cultured Drosophila melanogaster cells (Kc) was identified by its ability to be recognized, as an RNP, by anti-(U1)RNP antibodies from human lupus patients. Its sequence was deduced largely from direct analysis of the RNA molecule and then confirmed by DNA sequence determinations on a genomic clone isolated by hybridization to Drosophila U1 RNA. The Drosophila U1 RNA sequence exhibits 72% agreement with human U1 RNA. Nucleotides 3-11, which are complementary to the entire consensus sequence for donor (5') splice junctions in hnRNA, and to part of the acceptor (3') consensus, are exactly conserved. However, nucleotides 14-21, postulated to interact only with acceptor junctions, differ. Comparison of the Drosophila U1 sequence with vertebrate U1 sequences allows a particular secondary structure model to be preferred over others. These results are consistent with the hypothesis that U1 snRNPs are involved in splicing, but suggest specific modifications of the model detailing molecular interactions between U1 RNA and hnRNA during the splicing reaction.

Animals↗
PubMed1993

Enzymatic RNA synthesis with deoxynucleoside 5'-O-(1-thiotriphosphates).

We have investigated the incorporation of 2'-deoxynucleoside-5'-O-(1-thiotriphosphates) into RNA transcripts using T7 RNA polymerase. With the exception of [alpha-S]dGTP, we obtained full-length transcripts of pre-tRNA(Phe) and pre-tRNA(Tyr) using an appropriate mixture of 2'-deoxynucleoside 5'-O-(1-thiotriphosphate) and the corresponding normal nucleoside triphosphate. The yields of the transcripts were comparable to those obtained with unmodified NTPs. Both substrates, [alpha-S]dTTP and [alpha-S]dATP, were inserted specifically. However, [alpha-S]dCTP was excluded at specific sites. We could not obtain transcripts using the deoxyguanosine derivative.

Base Sequence↗
PubMed1999

Identification of a protein component of a mammalian tRNA(Sec) complex implicated in the decoding of UGA as selenocysteine.

This report describes a novel RNA-binding protein, SECp43, that associates specifically with mammalian selenocysteine tRNA (tRNA(Sec)). SECp43, identified from a degenerate PCR screen, is a highly conserved protein with two ribonucleoprotein-binding domains and a polar/acidic carboxy terminus. The protein and corresponding mRNA are generally expressed in rat tissues and mammalian cell lines. To gain insight into the biological role of SECp43, affinity-purified antibody was employed to identify its molecular partners. Surprisingly, the application of native HeLa cell extracts to a SECp43 antibody column results in the purification of a 90-nt RNA species identified by direct sequencing and Northern blot analysis as tRNA(Sec). The purification of tRNA(Sec) by the antibody column is striking, based on the low abundance of this tRNA species. Using recombinant SECp43 as a probe for interacting protein partners, we also identify a 48-kDa interacting protein, which is a possible component of the mammalian selenocysteine insertion (SECIS) pathway. To our knowledge, SECp43 is the first cloned protein demonstrated to associate specifically with eukaryotic tRNA(Sec).

Amino Acid Sequence↗
PubMed1992

A nucleosome core is transferred out of the path of a transcribing polymerase.

We have determined the fate of a nucleosome core on transcription. A nucleosome core was assembled on a short DNA fragment and ligated into a plasmid containing a promoter and terminators for SP6 RNA polymerase. The nucleosome core was stable in the absence of transcription. The distribution of nucleosome cores after transcription was examined. The histone octamer was displaced from its original site and reformed a nucleosome core at a new site within the same plasmid molecule, with some preference for the untranscribed region behind the promoter. These observations eliminate several models that have been proposed for transcription through a nucleosome core. Our results suggest that a nucleosome core in the path of a transcribing polymerase is displaced by transfer to the closest acceptor DNA.

Animals↗
PubMed1999

Mutations in conserved domain II of the large (L) subunit of the Sendai virus RNA polymerase abolish RNA synthesis.

The large (L) protein of Sendai virus complexes with the phosphoprotein (P) to form the active RNA-dependent RNA polymerase. The L protein is believed to be responsible for all of the catalytic activities of the polymerase associated with transcription and replication. Sequence alignment of the L proteins of negative-strand RNA viruses has revealed six conserved domains (I-VI) thought to be responsible for the enzymatic activities. Charged-to-alanine mutagenesis was carried out in a highly charged, conserved region (amino acids 533-569) within domain II to test the hypothesis of Müller et al. [J. Gen. Virol. 75, 1345-1352 (1994)] that this region may contribute to the template binding domain of the viral RNA polymerase. The mutant proteins were tested for expression and stability, the ability to synthesize viral RNA in vitro and in vivo, and protein-protein interactions. Five of the seven mutants were completely defective in all viral RNA synthesis, whereas two mutants showed significant levels of both mRNA and leader RNA synthesis. One of the transcriptionally active mutants also gave genome replication in vitro although not in vivo. The other mutant was defective in all the replication assays and thus the mutation uncoupled transcription and replication. Because the completely inactive L mutants can bind to the P protein to form the polymerase complex and the polymerases bind to the viral nucleocapsid template, these amino acids are essential for the activity of the L protein.

Amino Acid Sequence↗
PubMed2005

The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch.

Riboswitches are genetic control elements that usually reside in untranslated regions of messenger RNAs. These folded RNAs directly bind metabolites and undergo allosteric changes that modulate gene expression. A flavin mononucleotide (FMN)-dependent riboswitch from the ribDEAHT operon of Bacillus subtilis uses a transcription termination mechanism wherein formation of an RNA-FMN complex causes formation of an intrinsic terminator stem. We assessed the importance of RNA transcription speed and the kinetics of FMN binding to the nascent mRNA for riboswitch function. The riboswitch does not attain thermodynamic equilibrium with FMN before RNA polymerase needs to make a choice between continued transcription and transcription termination. Therefore, this riboswitch is kinetically driven, and functions more like a "molecular fuse." This reliance on the kinetics of ligand association and RNA polymerization speed might be common for riboswitches that utilize transcription termination mechanisms.

Bacillus subtilis↗