Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2'-groups using a mutantT7 RNA polymerase (RNAP). (1/547)

A T7 RNAP mutant (Y639F) which eliminates discrimination of the chemical character of the NTP ribose 2'-group, facilitates incorporation of non-canonicalsubstrates into nucleic acids. However, transcripts containing a high percentage of non-canonical NMPs are poorly extended due to effects of the 2'-substituents on the transcript:template hybrid conformation. We tested the addition of compounds that stabilize A-type helix geometry to the reaction. High concentrations of polyamines, together with other changes in reaction conditions, greatly increased the synthesis of transcripts heavily substituted with non-canonical ribose 2'-groups. Template structures that facilitate promoter opening increased the efficiency of reactions where non-canonical substrates were incorporated during transcription of +1 to +6.  (+info)

Comparison of synonymous codon distribution patterns of bacteriophage and host genomes. (2/547)

Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available.  (+info)

The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition. (3/547)

Bacteriophage T7 DNA primase recognizes 5'-GTC-3' in single-stranded DNA. The primase contains a single Cys4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys4 motif contacts cytosine of 5'-GTC-3' and may also contribute to thymine recognition. Residues His33 and Asp31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5'-GTC-3', by base pairing. Our deduced mechanism for recognition of ssDNA by Cys4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys2His2 zinc fingers.  (+info)

Co-expression of gene 31 and 23 products of bacteriophage T4. (4/547)

Folding of the major capsid protein of bacteriophage T4 encoded by gene 23 is aided by Escherichia coli GroEL chaperonin and phage co-chaperonin gp31. In the absence of gene product (gp) 31, aggregates of recombinant gp23 accumulate in the cell similar to inclusion bodies. These aggregates can be solubilized with 6 M urea. However, the protein cannot form regular structures in solution. A system of co-expression of gp31 and gp23 under the control of phage T7 promoter in E. coli cells has been constructed. Folding of entire-length gp23 (534 amino acid residues) in this system results in the correctly folded recombinant gp23, which forms long regular structures (polyheads) in the cell.  (+info)

The environment of 5S rRNA in the ribosome: cross-links to 23S rRNA from sites within helices II and III of the 5S molecule. (5/547)

Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7 transcription from synthetic DNA templates. The central fragment, comprising residues 33-71 of the molecule, was transcribed in the presence of 4-thiouridine triphosphate together with [32P]UTP. The three transcripts were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine residues at positions 40, 48, 55 and 65 in helices II and III. After ligation, the 4-thiouridine residues were derivatised with p -azidophenacyl bromide. The modified 5S rRNA was reconstituted into 50S subunits and these subunits were used to prepare 70S ribosomes in the presence or absence of tRNA and mRNA. The azidophenyl groups were then photoactivated by mild irradiation at 300 nm and the products of cross-linking analysed by our standard procedures. Multiple cross-links from 5S rRNA to two distinct regions of the 23S rRNA were observed. The first region was located in helix 38 in Domain II of the 23S molecule, with cross-links at sites between nucleotides 885 and 922. The second region covered helices 81-85 in Domain V, with sites between nucleotides 2272 and 2345. Taken together with previous data, these results serve to define the arrangement of the 5S rRNA molecule relative to the 23S rRNA within the 50S subunit.  (+info)

RNA polymerase-specific nucleosome disruption by transcription in vivo. (6/547)

The nucleosomal chromatin structure within genes is disrupted upon transcription by RNA polymerase II. To determine whether this disruption is caused by transcription per se as opposed to the RNA polymerase source, we engineered the yeast chromosomal HSP82 gene to be exclusively transcribed by bacteriophage T7 RNA polymerase in vivo. Interestingly, we found that a fraction of the T7-generated transcripts were 3' end processed and polyadenylated at or near the 3' ends of the hsp82 and the immediately downstream CIN2 genes. Surprisingly, the nucleosomal structure of the T7-transcribed hsp82 gene remained intact, in marked contrast to the disrupted structure generated by much weaker, basal level transcription of the wild type gene by RNA polymerase II under non-heat shock conditions. Therefore, disruption of chromatin structure by transcription is dependent on the RNA polymerase source. We propose that the observed RNA polymerase dependence for transcription-induced nucleosome disruption may be related either to the differential recruitment of chromatin remodeling complexes, the rates of histone octamer translocation and nucleosome reformation during polymerase traversal, and/or the degree of transient torsional stress generated by the elongating polymerase.  (+info)

Vaccinia virus-bacteriophage T7 expression vector for complementation analysis of late gene processes. (7/547)

A vaccinia virus-bacteriophage T7 RNA polymerase hybrid transient expression vector has been developed for complementation analysis of late gene functions in vaccinia virus. The conditionally defective virus ts21 was modified to express the bacteriophage T7 RNA polymerase. The derived virus, vtsT7, was conditionally defective in viral late gene expression but produced high levels of a target protein under the control of a T7 promoter at non-permissive temperatures. The level of beta-galactosidase expression under the control of a T7 promoter was slightly lower in vtsT7 infections than those with the prototypical T7 RNA polymerase vector vTF7.3. However, the levels of expression for the human immunodeficiency virus envelope gene, a protein which undergoes post-translational modification, was slightly higher in vtsT7 infections, suggesting that some proteins may be expressed better in the absence of vaccinia virus late gene expression. Infections using vtsT7 at a low m.o.i. at 39 degrees C resulted in the accumulation of high molecular mass, non-linear replicative intermediates of vaccinia virus DNA replication and high levels of expression of a transfected gene proximal to a T7 promoter. The virus vtsT7 provides a means for the analysis of potential trans-acting factors participating in vaccinia virus late processes such as resolution of DNA replicative intermediates.  (+info)

Preparation of HIV TAR RNA with RNA scissors. (8/547)

Two hammerhead ribozymes derived from plant pathogenic RNAs were used to cut off the HIV TAR RNA from the T7 RNA transcript through a cis cleavage reaction. Stem I of the (+)vLTSV ribozyme comprises 8 nucleotides of the 5' terminus of TAR RNA, but stem III of the (+)sTRSV ribozyme consists of 8 nucleotides of the 3' end of TAR RNA. The construct containing two GUC hammerhead ribozyme target sequences identified the cleavage sites to cut off a required RNA molecule. This method was applied for preparation of 35 nt long TAR RNA. Its activity was proved by the complex formation with the Tat protein. It seems that this approach based on RNA scissors can also be used for the generation of required RNA molecules, RNA decoys or RNA aptamers in vivo.  (+info)

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