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PubMed2003

Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus.

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.

Amino Acid Sequence↗
PubMed2006

An inducible T7 RNA polymerase-dependent plasmid system.

RNA interference (RNAi) has become a powerful tool for the specific silencing of gene transcription. Especially the targeting of genes in mammalian cells has been greatly improved by generating plasmid based and viral vector-based systems. This permits expression of short hairpin RNA (shRNA) on a longterm basis. However, an inducible expression of shRNA is required, if the target is essential for cell survival. We developed a doxycycline-inducible two-plasmid system for the expression of a ribozyme-processed shRNA. In contrast to other existing systems, we use the highly specific T7 phage RNA polymerase, which does not interact with cellular factors; therefore, interference with cellular functions is limited. One plasmid is responsible for doxycycline-dependent expression of T7 RNA polymerase and a second plasmid expresses a ribozyme-processed shRNA under the control of a T7 promoter. Our results showed that doxycycline- dependent expression of T7 RNA polymerase was tightly controlled and expression of an shRNA against firefly luciferase inhibited 86% of luciferase activity. In conclusion, our plasmid system provides a very useful tool for analyzing essential gene functions in vitro.

Base Sequence↗
PubMed1990

Effect of 5' splice site mutations on splicing of the preceding intron.

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

Adenoviruses, Human↗
PubMed1998

Cellular protein hnRNP-A1 interacts with the 3'-end and the intergenic sequence of mouse hepatitis virus negative-strand RNA to form a ribonucleoprotein complex.

We previously showed that several cellular proteins specifically bind to the 3'-end and the intergenic sequences of the negative-strand RNA of mouse hepatitis virus (MHV), and proposed that these distant RNA sequences can be brought together by cellular and viral proteins (Furuya and Lai, 1993; Zhang et al., 1994; Zhang and Lai, 1995). The cellular protein p35 has been identified as a heterogeneous nuclear ribonucleoprotein (hnRNP) A1. We have now expressed hnRNP-A1 as a glutathione-S-transferase (GST) fusion protein and demonstrated that the amino terminal two-thirds of hnRNP-A1 interacted with the two MHV regulatory RNA sequences (3'-end and intergenic sequences) through protein-RNA interaction while its carboxy-terminal glycine-rich domain mediated homomeric (protein-protein) interactions. In a partially reconstituted reaction, in which the two MHV RNA fragments and the purified GST-hnRNP-A1 fusion protein were mixed, an RNP complex was formed. Depletion of either hnRNP-A1 or one of the RNA components abolished the complex formation. These results indicate that hnRNP-A1 can mediate the formation of an MHV RNP complex, which includes both the negative-strand leader and intergenic sequences. Site-directed mutagenesis revealed that mutations in the MHV intergenic sequences, which inhibited MHV RNA transcription, also inhibited the RNP complex formation. Deletion analysis showed that the amino terminal RNA-binding domains of hnRNP-A1 is essential for the RNP complex formation while the carboxy-terminal protein-binding domain enhanced the complex formation by 90-fold. These findings provide direct evidence demonstrating that the negative-strand leader RNA and intergenic sequences can form an RNP complex mediated by cellular protein hnRNP-A1.

Animals↗
PubMed2002

Sequence analysis of the mouse IRBP gene and cDNA.

PURPOSE: To understand the structure of the mouse interphotoreceptor retinoid-binding protein (IRBP) gene and to compare the predicted primary structure within each repeat of IRBP with its relatives. To compare the levels of expression of IRBP RNA in normal and knockout mice. METHODS: The DNA sequence was determined by sub-cloning restriction fragments of the IRBP gene from 129/Sv P1 clones. Primers were designed to utilize the walking approach. Additional sequences were obtained by PCR amplification from genomic DNA and direct sequencing of products. Mouse retina RNA was subjected to reverse transcription coupled to PCR and the accumulation of double stranded DNA product was monitored with SYBR Green. The PCR primers flanked Intron C, to avoid the analysis of contaminating genomic DNA. RESULTS: Altogether a contig was assembled with a final length of about 14.4 kb. The mouse gene structure is similar to the pattern of exons and introns in the bovine and human genes, with a long first exon encoding most of the protein. The splice site boundaries closely match consensus sequences and the exons appear to be identically placed among the three species (bovine, human, and mouse). A region containing a repeated sequence of low complexity is located about 1.75 to 1.4 kb upstream of the transcription start site. A second region containing another low complexity repeat is found in Intron C close to the end of Exon 3. A limited number of weak consensus polyadenylation signals in the 3' region suggest at least three different transcription terminators that apparently give rise to the previously known mouse IRBP mRNAs. The mRNA for IRBP was detected in normal and part of the mRNA was detected in the IRBP knockout mouse, consistent with previous observations. The level of the IRBP mRNA remnant was reduced about 10 fold in the knockout mouse, also consistent with the previously reported absence of Repeat 4 immunoreactivity. CONCLUSIONS: The strong conservation in intron-exon positions, gene structure, and protein sequence among mammals supports an important biological role for these signals and for the IRBP protein in vision. Low levels of aberrant IRBP mRNA in the knockout mouse are consistent with no immunologically detectable Repeat 4 protein in this mouse.

Amino Acid Sequence↗
PubMed2001

Functional analysis of PA binding by influenza a virus PB1: effects on polymerase activity and viral infectivity.

Influenza A virus expresses three viral polymerase (P) subunits-PB1, PB2, and PA-all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain alpha, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain alpha. The role of the minimal PB1 domain alpha in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

Amino Acid Sequence↗
PubMed2000

In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase.

Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem-loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT-PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.

Base Sequence↗
PubMed2001

Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations.

Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).

Base Sequence↗
PubMed1988

RNA synthesis: strategies for the use of bacteriophage RNA polymerases.

This communication presents an overview of the methods for the synthesis of RNA with virtually any desired sequence. The use of transcription vectors is a powerful and convenient approach, if the cloned gene of interest has restriction sites at the proper positions. To overcome these limitations, two methods were developed where chemically synthesized oligodeoxynucleotides (oligos) were applied to define the 3' and 5' termini of the chosen transcripts. Both approaches use cloned genes and the template DNA is synthesized with DNA polymerase I (Klenow fragment). Consequently, there are no size limitations for the synthesized RNAs. For short transcripts, the entire template DNA (including the promoter sequence) can be synthesized chemically and any desired RNA sequence is possible. Recently, it was shown that even oligos without any promoter sequence can be used as template DNA for RNA polymerases. Experimental data are presented for two approaches. The first example is the synthesis of template DNA for T7 RNA polymerase where two oligos (initiator and terminator) define the beginning and end of transcripts from a cloned gene. The second example is the use of simple oligos as templates for RNA polymerases. The major problem encountered was the inaccurate transcription termination, which resulted in one or two additional nucleotides beyond the encoded sequence.

Base Sequence↗
PubMed2000

Isolation and characterisation of a chick cDNA encoding the RNA polymerase common subunit RPB6.

The RPB6 cDNA of chicken, encoding one of the small subunits common to all three nuclear DNA-dependent RNA polymerases, has been isolated from an expression cDNA library by screening with a differential display derived probe, representing a gene shown to be highly up-regulated in early heart development. The nucleotide sequence of the cDNA isolated predicts a protein sequence of 127 amino acids. This sequence shares 124 amino acids (98% homology) with the human RNA polymerase II subunit 14.4 kDa (RPB6) and hamster hRPB6 and 123 amino acids (97% homology) with Rattus norvegicus RNA polymerase II subunit RPB6. Other conserved motifs in this protein and potential functions of RPB6 are discussed.

Amino Acid Sequence↗
PubMed1988

Location of sequences in the nar promoter of Escherichia coli required for regulation by Fnr and NarL.

Nitrate reductase, encoded by the nar operon in Escherichia coli, is produced only under anaerobic conditions and induced to its maximum level in the presence of nitrate. The anaerobic expression of the nar operon depends on the fnr gene product (Fnr), and the stimulation of anaerobic expression by nitrate requires the narL gene product (NarL). Distinct regulatory domains within the nar promoter are involved in these two responses. The specific locations of the sequences required for these two regulatory mechanisms were identified by analysis of a detailed set of deletions extending into the regulatory region of the nar operon from the 5' end. A region located around -55 base pairs (bp) from the transcriptional start site and immediately upstream from the presumed RNA polymerase binding site was required for the response to Fnr and anaerobic conditions. A base sequence no longer than 27 bp, located at about -200 bp, was essential for the stimulation by nitrate coupled with NarL. This NarL-specific sequence was equally effective if positioned 10 or 11 bp further upstream or downstream from its wild type position. However, it was ineffective if positioned 4, 6, or 14 bp or greater distances either upstream or downstream. Apparent autoregulation by active nitrate reductase occurred in all 5'-deletion constructions which retained the Fnr response, indicating that this regulatory phenomenon involves sequences located no further than -64 bp from the transcription start site.

Anaerobiosis↗
PubMed1999

Transcriptional organization and in vivo role of the Escherichia coli rsd gene, encoding the regulator of RNA polymerase sigma D.

The regulator of sigma D (Rsd) was identified as an RNA polymerase sigma70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of sigma70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953-4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, sigmaS-dependent P1 and sigma70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the sigma70- or sigmaS-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the sigma70- and sigmaS-dependent gene expression was increased or decreased, respectively. On the other hand, the sigma70- or sigmaS-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the sigmaS-dependent transcription in the rsd mutant is overcome by increased production of the sigmaS subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase sigma subunit from sigma70 to sigmaS during the transition from exponential growth to the stationary phase.

Bacterial Proteins↗
PubMed1987

[Highly selective affinity labeling of a promoter in a complex with E. coli RNA-polymerase by alkylating derivatives of initiating substrates].

The complex [promoter A2 X E. coli RNA polymerase] was treated with phosphoamides, derivatives of 4-[N-methyl, N-(2-chloroethyl)]-aminobenzylamine and guanosine-5'-mono-, di-, and triphosphates with the alkylating group attached to the terminal phosphates. After this, [alpha-32P]CTP was added. Residues of the affinity reagents bound covalently at the first stage were elongated by radioactive -pC residues due to the catalytic action of the active centre of RNA polymerase. Affinity labelled were beta-and sigma-subunits of the enzyme, and the promoter. The affinity label was localized on -pGpC residues. A guanine residue was alkylated in the promoter as suggested by radioactivity elimination kinetics. As the data obtained and the previously known length of the reagent (maximum distance between the alpha-phosphorus atom of the reagent and the point of alkylation is less than 0.6 nm) indicate, there is a direct rather than protein-mediated contact between the template and the substrate within the complex [promoter X RNA polymerase].

Affinity Labels↗
PubMed1988

Sequences in the 5' proximal segment of the paused transcript affect NusA-mediated enhancement of transcriptional pausing.

NusA protein is a transcription elongation and termination factor that acts to enhance pausing of RNA chain growth by RNA polymerase at specific sites on DNA templates. We demonstrate that this enhancement of pausing in tR1, the transcription termination site between genes cro and cII of phage lambda, is inhibited by DNA oligonucleotides complementary to a segment of the nascent RNA just preceding the sequence that is thought to be a part of the stem of an RNA hairpin that is responsible for pausing, but not by oligonucleotides complementary to segments containing the boxA sequence or the sequences up to 40 nucleotides upstream from boxA. The results are consistent with a model proposed by Landick and Yanofsky (Landick, R., and Yanofsky, C. (1987) J. Mol. Biol. 196, 363-377) in which the NusA protein acting as a subunit of RNA polymerase in the elongation complex contacts a structure near the 3' end of the nascent transcript and uses the binding energy to shift the equilibrium between elongation and paused conformational isomers of the transcription complex in favor of the paused isomer.

Bacterial Proteins↗
PubMed1993

Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences.

Small guide RNAs (gRNAs) may direct RNA editing in kinetoplastid mitochondria. We have characterized multiple gRNA genes from Trypanosoma brucei (EATRO 164), that can specify up to 30% of the editing of the COIII, ND7, ND8, and A6 mRNAs and we have also found that the non-translated region of edited COIII mRNA of strain (EATRO 164) differs from that of another strain. Several of the gRNAs specify overlapping regions of the same mRNA often specifying sequence beyond that required for an anchor duplex with the next gRNA. Some gRNAs have different sequence but specify identical editing of the same region of mRNA. These data indicate a complex gRNA population and consequent complex pattern of editing in T. brucei.

Animals↗
PubMed1987

Specific activation of transcription initiation by the sequence-specific DNA-binding agents distamycin A and netropsin.

A series of promoters with nine base-pair substitutions in the spacer DNA separating the -10 and -35 regions was used to demonstrate that Escherichia coli RNA polymerase is sensitive to events affecting the spacer DNA--a region not directly contacted by the enzyme. The drugs distamycin A and netropsin specifically enhanced the rate of functional complex formation at a promoter bearing a substitution of nonalternating A-T base pairs. The effect is exerted at an early step in the RNA polymerase-promoter interaction. We hypothesize that a drug-induced structural alteration in the spacer DNA occurs, similar to that normally resulting from RNA polymerase binding. These findings are relevant to an understanding of potential mechanisms of transcription activation.

Base Sequence↗
PubMed2004

The crystal structure of the monomeric reverse transcriptase from Moloney murine leukemia virus.

Reverse transcriptases (RTs) are multidomain enzymes of variable architecture that couple both RNA- and DNA-directed DNA polymerase activities with an RNase H activity specific for an RNA:DNA hybrid in order to replicate the single-stranded RNA genome of the retrovirus. Previous structural work has been reported for the heterodimeric HIV-1 and HIV-2 RTs. We now report the first crystal structure of the full-length Moloney murine leukemia virus (MMLV) RT at 3.0 A resolution. The structure reveals a clamp-shaped molecule resulting from the relative positions of the thumb, connection, and RNase H domains that is strikingly different from the HIV-1 RT and provides the first example of a monomeric reverse transcriptase. A comparative analysis with related DNA polymerases suggests a unique trajectory for the template-primer exiting the polymerase active site and provides insights regarding processive DNA synthesis by MMLV RT.

Amino Acid Sequence↗
PubMed1989

Characterization of a new prokaryotic transcriptional activator and its DNA recognition site.

The expression of the Bacillus subtilis phage phi 29 DNA is controlled by the viral gene 4 product, which is required for the initiation of transcription at the unique late promoter A3. Protein p4 binds specifically to a phi 29 DNA fragment containing the A3 promoter. DNase I footprinting analysis has shown that the DNA binding region for protein p4 is located between nucleotides -50 and -100 relative to the transcription start site. Methylation interference assays suggest that two eight base-pair long inverted repeats located within this binding region are the protein p4 recognition sequence. These results, together with the fact that the protein p4-dependent in vitro transcription requires the B. subtilis sigma 43-RNA polymerase, indicate that protein p4 is a transcriptional activator. The protein p4 DNA recognition region is statically bent as suggested by gel retardation and chemical cleavage assays. A model of protein p4 binding to its DNA target site is proposed.

Bacillus subtilis↗