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At least 451 records · Page 25Linked to original sources
PubMed1990

Retron for the 67-base multicopy single-stranded DNA from Escherichia coli: a potential transposable element encoding both reverse transcriptase and Dam methylase functions.

The region (retron-Ec67) required for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec67) from a clinical isolate of Escherichia coli was mapped at a position equivalent to 19 min on the K-12 chromosome. The element containing the retron consisted of a unique 34-kilobase sequence that was flanked by direct repeats of a 26-base-pair sequence found in the K-12 chromosomal DNA. This suggests that the 34-kilobase element was probably integrated into the E. coli genome by a mechanism related to transposition or phage integration. In the 34-kilobase sequence an open reading frame of 285 residues was found, which displays 44% sequence identity with the E. coli Dam methylase. Interestingly, there are three GATC sequences, the site of Dam methylation, in the promoter region of the gene for reverse transcriptase.

Amino Acid Sequence↗
PubMed2006

Azobenzene-tethered T7 promoter for efficient photoregulation of transcription.

Azobenzene was additionally introduced into side chain of T7 promoter for the photocontrol of transcription reaction by T7 RNA polymerase (T7 RNAP). When a single azobenzene molecule was introduced into the T7 promoter either at the loop-binding region of the RNAP (-7 to -11 position) or at the unwinding region (-1 to -4 position), transcription was suppressed in the trans-form but proceeded faster in the cis-form. The amount of transcripts after UV irradiation with respect to that in the dark was 1.5-2.0-fold. Kinetic analysis of the transcription reaction revealed that the photoregulatory mechanism was different in these positions. The photoisomerization of an azobenzene at the loop-binding region primarily affected Km. On the other hand, the isomerization of an azobenzene at the unwinding region mainly affected kcat. Still more clear-cut photoregulation was achieved when two azobenzenes were introduced into both loop-binding and unwinding regions, respectively: transcription proceeded 7.6-fold faster after UV irradiation than that in the dark. This synergistic effect was observed only when two azobenzenes were introduced into these two different regions, respectively, and introduction of them into the same loop-binding region drastically lowered the transcription activity. The cooperation of two azobenzenes at loop-binding and unwinding regions would contribute to the clear-cut photoregulation of transcription.

Azo Compounds↗
PubMed1997

RNA splicing ligase activity in the archaeon Haloferax volcanii.

At least two separate enzymes, an endonuclease and a ligase, appear to be involved in tRNA splicing in halophilic archaea. We have identified and partially characterized a splicing ligase activity in cell extracts of Haloferax volcanii that can ligate deproteinized exon products generated in a separate endonuclease reaction. As in vitro transcribed partial intron-deleted derivative of H. volcanii elongator tRNA(Met) is used as substrate for the endonuclease. The ligase can also join the two exons that are independently eluted from the gels. This ligase activity is observed at a range (50 mM to 2.8 M) of monovalent cations in the assays, but is abolished when the enzyme preparations are depleted of the monovalent cations. In contrast, H. volcanii splicing endonuclease has been reported to require divalent cations and is inhibited by monovalent cations. Our endonuclease assays confirm these reports, and also show that the endonuclease is not permanently inactivated even in high monovalent cation containing extracts. The ligase activity in the extracts does not appear to require any divalent cation or exogenously added source of energy or phosphate.

Archaea↗
PubMed1994

Determination of intrinsic transcription termination efficiency by RNA polymerase elongation rate.

Transcription terminators recognized by several RNA polymerases include a DNA segment encoding uridine-rich RNA and, for bacterial RNA polymerase, a hairpin loop located immediately upstream. Here, mutationally altered Escherichia coli RNA polymerase enzymes that have different termination efficiencies were used to show that the extent of transcription through the uridine-rich encoding segment is controlled by the substrate concentration of nucleoside triphosphate. This result implies that the rate of elongation determines the probability of transcript release. Moreover, the position of release sites suggests an important spatial relation between the RNA hairpin and the boundary of the terminator.

Base Sequence↗
PubMed1991

Abortive products as initiating nucleotides during transcription by T7 RNA polymerase.

The kinetics of formation of abortive initiation products during transcription of a synthetic template (encoding the transcript GAUGGC) by T7 RNA polymerase have been determined. This study revealed that while total RNA was formed in the reaction as expected, the levels of the dinucleoside tetraphosphate guanylyl-3',5'-adenosine-5'-triphosphate (pppGpA) and trinucleoside pentaphosphate guanylyl-3',5'-adenosine-3',5'-uridine-5'-triphosphate (pppGpApU) formed by premature termination of transcription reached a maximum after 10 min, and then decreased. Transcription of the same template, in the presence of either [gamma-32P]GTP and ATP, or GTP and [alpha-32P]ATP, gave the 32P-labeled dinucleotides *pppGpA and pppG*pA. Incorporation of each of these substrates into longer RNA transcripts in the same enzyme-template system was demonstrated. The incorporation was shown to require the presence of template in the reaction mixture. The requirement for base complementarity restricts the position of incorporation to that of initiating (5') nucleotide. Transcription of a second template, which encodes an RNA transcript having the partial sequence GpA at two internal positions, in the presence of each of the labeled dinucleoside tetraphosphates, failed to bring about the synthesis of significant yields of any longer radiolabeled transcripts. It is concluded that dinucleoside tetraphosphate (and perhaps trinucleoside pentaphosphate) can function as initiating nucleotides when complementary to the nucleotide sequence at promoter regions. However, a dinucleotide is not used as substrate for subsequent chain elongation in T7 RNA polymerase catalyzed transcription reactions.

Base Sequence↗
PubMed1993

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase.

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.

Amino Acid Sequence↗
PubMedDate not supplied

[Alternative transcripts from POLRMT responsible for synthesis of nuclear RNA polymerase IV].

By analyzing ESTs that correspond to human POLRMT gene encoding mitochondrial RNA polymerase (mtRNAP) we revealed an alternatively spliced transcript. We confirmed the existence of the transcript that contain additional 225 nucleotides from proximal part of intron 1 by RT-PCR using RNA from HeLa cells. In mouse and rat there are similar alternative transcripts that contain entire intron 1 sequences. In addition, in mouse we revealed third transcript that contain extra exon derived from 142 bp of intron 2. The revealed alternative transcripts, in contrast to the mRNA encoding mtRNAP, specify N-terminally truncated protein lacking mitochondrial targeting signal. This protein has strictly nuclear localization and corresponds to nuclear RNA polymerase IV that we recently identified.

3T3 Cells↗
PubMed1991

The nut site of bacteriophage lambda is made of RNA and is bound by transcription antitermination factors on the surface of RNA polymerase.

The boxA and boxB components of the lambda nut site are important for transcriptional antitermination by the phage N protein. We show here that boxA and boxB RNA in N-modified transcription complexes are inaccessible to ribonucleases and have altered sensitivity to dimethylsulfate. N and NusA suffice to weakly protect boxB, independently of boxA and other factors. However, efficient protection of the entire nut site from ribonucleases requires boxA and boxB, N, NusA, NusB, S10, and NusG. Mutations in RNA polymerase, which inhibit antitermination by N in vivo, disallow protection of the nut site during transcription in vitro; therefore, the surface of RNA polymerase must coordinate the formation of complexes containing the antitermination factors and nut site RNA.

Bacteriophage lambda↗
PubMed1991

RNA polymerase activity may regulate transcription initiation and attenuation in the rplKAJLrpoBC operon in Escherichia coli.

The relationship between global RNA transcription capacity and transcript initiation, attenuation, and stability in the rplKAJLrpoBC operon of Escherichia coli has been examined. The rplKAJLrpoBC operon encodes in order the four large ribosome subunit proteins, L11, L1, L10, and L12, and the two large beta and beta' subunits of RNA polymerase. Operon transcripts are initiated at two promoters, PL11 and PL10. The L12-beta intergenic space contains a transcription attenuator which, during balanced growth, terminates about 80% of the transcripts exiting the L12 gene; the remaining transcripts read through into the beta and beta' encoding genes. The capacity for global transcription initiation was modulated using a strain carrying a temperature-sensitive, initiation-defective mutation in rpoC. Following a shift to 39 degrees C, the global transcription initiation capacity was reduced to about one-half the level at 30 degrees C. This partial restriction resulted in a decrease in the stability of distal beta mRNA, whereas the stability of proximal L11-L1 and L10-L12 mRNA was not changed. Measurements of the synthesis rates of L11-L1, L10-L12, and beta mRNAs relative to total RNA synthesis indicated that this operon was selectively transcribed when the initiation capacity of RNA polymerase was limited. The synthesis rates of L11-L1 and L10-L12 mRNA increased about 2-fold, whereas the synthesis rate of beta mRNA increased nearly 5-fold. The relative transcription of other ribosome component genes and the alpha subunit gene exhibited only a modest increase during the partial restriction. Protection from S1 nuclease was used to demonstrate that the preferential transcription within the operon of beta mRNA was the consequence of active regulation of termination-antitermination at the attenuator structure in the L12-beta intergenic space. These results demonstrate that global transcription capacity may be an important parameter in determining both initiation and attenuation of transcription of the rplKAJLrpoBC ribosomal protein-RNA polymerase operon.

Base Sequence↗
PubMed1995

Target sequence-specific inhibition of HIV-1 replication by ribozymes directed to tat RNA.

The structural motif formed between a hammerhead ribozyme and its substrate consists of three RNA double helices in which the sequence 5' to the XUY is termed helix I and the sequence 3' to the XUY helix III. Two hammerhead ribozymes targeted to the tat gene of HIV-1SF2 were designed to study target specificity and the potential effect of helix I mismatch on ribozyme efficacy both in vitro and in vivo. The first ribozyme (Rz1) targeted to the 5' splicing region of the tat gene was designed to cleave GUC*A. In HIV-1IIIB the A is changed to a G. The second ribozyme (Rz2) was targeted to the translational initiation region of the tat gene which is highly conserved among a variety of HIV-1 isolates, including both HIV-1SF2 and HIV-1IIIB. In vitro cleavage studies demonstrated that Rz1 efficiency cleaved HIV-1SF2 substrate RNA, but not HIV-1IIIB, presumably due to the base change from A to G. In contrast, Rz2 cleaved HIV-1SF2 or HIV-1IIIB substrate with equal efficiency. Both ribozymes were cloned into the 3' untranslated region of the neomycin gene (neo) within the pSV2neo vector and transfected into the SupT1 human CD4+ T cell line. Following selection, stable transfectants were challenged with either HIV-1SF2 or HIV-1IIIB virus. While Rz1-expressing cells were significantly protected from HIV-1SF2 infection, they exhibited no protection when infected with HIV-1IIIB virus. In contrast, Rz2 was effective in inhibiting the replication of both HIV-1SF2 and HIV-1IIIB in SupT1 cells. Expression of both ribozymes in these cells was demonstrated by Northern analysis. RT-PCR sequencing analysis confirmed the respective HIV-1 target sequence integrity. These data demonstrate the importance of the first base pair distal to the XUY within helix I of the hammerhead structure for both in vitro and in vivo ribozyme activities and imply that the effectiveness of the anti-HIV-1 ribozymes against appropriate target sequences is due to their catalytic activities rather than any antisense effect.

Base Sequence↗
PubMed1996

A SELEX primer.

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Avian Myeloblastosis Virus↗
PubMed1998

Effects of mutations in the C-terminal region of NIa protease on cis-cleavage between NIa and NIb.

Mutational analyses were carried out to investigate whether the nuclear inclusion protein a (NIa) C-terminal amino acids of turnip mosaic potyvirus play any roles in the cis-cleavage between NIa and NIb. The processing rate of the NIa-NIb junction sequence was decreased significantly by either V240D or Q243A mutation while little affected by F226D, V228E, K230E, I232D, or L235D mutation. The mutation of W212S, G213S, or I217D abolishing the cleavage at the NIb-CP or 6K1-cylindrical inclusion protein junction sequence decreased the processing rate to half the level of that of the wild type. Deletion of the C-terminal one (K230), two (S229 and K230), three (S229 to L231), or six amino acids (S229 to D234) as well as the insertion of five glycines between S229 and K230 or between S220 and Q221 did not affect significantly the cleavage while the deletion of 20 amino acids (Q218 to S237) decreased the processing rate to 73% of that of the wild type. These results rule out the possibility that the C-terminal region plays a role as a spacer in right placement of the NIa-NIb junction sequence and demonstrate that the C-terminal 20 amino acids from Q218 to S237 are not crucial for the cis-cleavage of the NIa-NIb junction sequence.

Amino Acid Sequence↗
PubMed1996

Sequence of the 3'-terminal region of a Zimbabwe isolate of cowpea aphid-borne mosaic virus (CABMV).

The 3'-terminal 1221 nucleotides of a Zimbabwe isolate of cowpea aphid-borne mosaic potyvirus (CABMV) genome have been sequenced. The sequence comprises an open reading frame (ORF) of 990 nucleotides and a 3' non-coding-region of 231 nucleotides followed by a poly-A. The ORF has high similarity to NIb and coat proteins (CP) of potyviruses. A potential CP Q/S cleavage site was identified, yielding a CP of 30.5 kDa containing 275 amino acids. The CABMV sequence is closely related to that of South African passiflora virus (SAPV) which should therefore be regarded as a strain of CABMV.

Amino Acid Sequence↗
PubMed1995

Analysis of the phylogenetic placement of different spumaretroviral genes reveals complex pattern of foamy virus evolution.

Foamy or spumaviruses are complex retroviruses. Phylogenetic trees have been constructed previously for either the polymerase or integrase domains showed a clustering of the foamy viruses relatively distant from other retroviruses. The most related retrovirus was found to be murine leukemia virus, irrespective of the method used or foamy viral gene analyzed. We analyze bel genes of different foamy viruses and compared the corresponding phylogenetic trees with those obtained from the pol genes that were constructed with refined computer programs. In addition, the nucleocapsid protein sequence of foamy viruses was used for a comparative phylogenetic analysis. Known biological properties of the individual FV protein domains are discussed to ascertain the apparent phylogenetic relatedness.

Amino Acid Sequence↗