Physical mapping of hybrid bacteriophage T7/T3 RNA polymerase genes (original) (raw)
Related papers
Journal of Molecular Biology, 1990
The bacteriophage T3 and T7 RNA polymerases are closely related, yet are highly specific for their own promoter sequences. To understand the basis of this specificity, T7 promoter variants that contain substitutions of T3-specific base-pairs at one or more positions within the T7 promoter consensus sequence were synthesized and cloned. Template competition assays between variant and consensus promoters demonstrate that the primary determinants of promoter specificity are located in the region from-10 to-12, and that the base-pair at-11 is of particular importance. Changing this base-pair from G" C, which is normally present in T7 promoters, to C'G, which is found at this position in T3 promoters, prevented utilization by the T7 RNA polymerase and simultaneously enabled transcription from the variant T7 promoter by the T3 enzyme. Substitution of T7 base-pairs with T3 base-pairs at other positions where the two consensus sequences diverge affected the overall efficiency with which the variant promoter was utilized by the T7 RNA polymerase, but these changes were not sufficient to permit recognition by the T3 RNA polymerase. Switching the-11 base-pair in the T3 promoter consensus to the T7 base-pair prevented utilization by the T3 RNA polymerase, but did not allow the T3 variant promoter to be utilized by the T7 RNA polymerase. This probably reflects a greater specificity of the T7 RNA polymerase ~ for base-pairs at other positions where the promoter sequences differ, most notably at-15. The magnitude of the effects of base substitutions in the T7 promoter on promoter strength (-llC >>-10C >-12A) correlates with the affinity of the T7 polymerase for the promoter variants, suggesting that the discrimination of the phage RNA polymerases for their promoters is mediated primarily at the level of DNA binding, rather than at the level of initiation.
Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters
Journal of Biological Chemistry, 1991
The specificity and structural simplicity of the bacteriophage T3, T7, and SP6 RNA polymerases make these enzymes particularly well suited for studies of polymerase-promoter interactions. To understand the initial recognition process between the enzyme and its promoters, DNA fragments that carry phage promoters were chemically modified by three different methods: base methylation, phosphate ethylation, and base removal. The positions at which these modifications prevented or enhanced binding by the RNA polymerases were then determined. The results indicate that specific contacts within the major groove of the promoter between positions-5 and-12 are important for phage polymerase binding. Removal of individual bases from either strand of the initiation region (-5 to +3) resulted in enhanced binding of the polymerase, suggesting that disruption of the helix in this region may play a role in stabilization of the polymerasepromoter complexes.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1993
Using a rifampicin-resistant RNA polymerase with altered specificity to different promoters, the D promoter of T7 phage DNA with increased affinity to the mutant enzyme was chosen. This promoter and the T7 A1 promoter with unchanged affinity as well as some nonpromoter DNA fragments were used to compare temperature-induced conformational transitions of RNA polymerase in the course of complex formation. Conformational alterations of RNA polymerase were monitored by the fluorescent label method. It was shown that RNA polymerase undergoes a set of conformational transitions during complex formation with each promoter, some of which were similar by the character of change to spectral parameters of the label (reflecting RP i and, probably, RP o formation). The local structure of complexes formed above 33°C differs for A1 and D. The conformational analysis reveals at least one temperature-dependent stage upon nonspecific interaction of the enzyme with nonpromoter DNA at 13-16°C. Models of functional organization of the enzyme recognizing center and some features of the structure of the promoters which may be essential for their recognition are discussed.
Proceedings of The National Academy of Sciences, 1985
The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the X PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.
Journal of Biological Chemistry, 1974
Further studies on the specificity of RNA chain initiation by T34nduced RNA polymerase have been carried out. The nucleotide penultimate to the 5'-guanosine triphosphate end of RNA chains has been determined. GMP is found exclusively in this second position of a growing RNA chain. The "PPi exchange reaction with ribonucleoside triphosphates catalyzed by T3 RNA polymerase on native T3 DNA template was also studied as a measure of the initiation reaction. It has been found that T3 RNA polymerase carries out an active exchange of 3*PPi into ATP in the presence of GTP. In contrast, in reactions containing GTP alone or with GTP + CTP or GTP + UTP, an active exchange of 32PPi only occurs into GTP. NO '*PPi incorporation was detected into either CTP or UTP. These results suggest that all RNA chains formed in T3 RNA polymerase reaction directed by native T3 DNA as template contain the sequence pppGp(G~)~(ApL. .. (where n and m = 1 or more) at the 5' terminus, indicating a high degree of specificity of initiation of RNA chains in this system. Infection of Escherichia coli with phage T3 leads to the induction of a new DNA-dependent RNA polymerase (l-5). The phage coded enzyme is physically and biochemically distinct from the E. coli RNA polymerase, and is required for expression of late regions of the T3 genome (l-7). One striking property of the T3 RNA polymerase (as well as the T7-induced RNA polymerase (8)) is its remarkable template specificity. T3-induced RNA polymerase will transcribe only native T3 DNA, and is ineffective with a wide variety of native DNAs tested so far. These include DNAs from bacteriophage T2 and T4; calf
Proceedings of the National Academy of Sciences of the United States of America, 1979
A transducing phage has been isolated which codes for the a subunit of Escherichia coli RNA polymerase. Transducing phage were selected from E. coli shotgun collections of HindIII or Sac I fragments cloned into Charon 25, a new bacteriophage X vector that is capable of forming Iyosogens at high temperature. Transduction of an E. coli strain carrying a temperature-sensitive mutation in the a gene was used for the selection. The positions of restriction sites for Sac I, HindIII, Xho I, Bgl II, and Kpn I in the cloned bacterial DNA segments were determined. Phage containing the HindIII fragment complement both primase (dnaG) and o (rpoD) whereas those containing the Sac I fragment complement only a. Results of analyses of the proteins made both in vivo after infection of UV-irradiated cells and in vitro in a coupled transcription/ translation system suggest that a Sac I site separates the promoter for a from the a structural gene. The direction of transcription of a was determined to be clockwise with respect to the E. coli genetic map. Escherichia coli RNA polymerase is a multisubunit enzyme composed of a, 13, f3', and oa subunits. The enzyme is found in two forms: as holoenzyme (a2/3f'), capable of selective DNA
Journal of Molecular Biology, 2000
We show here that transcription by the bacteriophage T7 RNA polymerase increases the deamination of cytosine bases in the non-transcribed strand to uracil, causing C to T mutations in that strand. Under optimal conditions, the mutation frequency increases about ®vefold over background, and is similar to that seen with the Escherichia coli RNA polymerase. Further, we found that a mutant T7 RNA polymerase with a slower rate of elongation caused more cytosine deaminations than its wild-type parent. These results suggest that promoting cytosine deamination in the non-transcribed strand is a general property of transcription in E. coli and is dependent on the length of time the transcription bubble stays open during elongation. To see if transcription-induced mutations have in¯uenced the evolution of bacteriophage T7, we analyzed its genome for a bias in base composition. Our analysis showed a signi®cant excess of thymine over cytosine bases in the highly transcribed regions of the genome. Moreover, the average value of this bias correlated well with the levels of transcription of different genomic regions. Our results indicate that transcription-induced mutations have altered the composition of bacteriophage T7 genome and suggest that this may be a signi®cant force in genome evolution.
Journal of Bacteriology, 1985
Highly efficient promoters of coliphage T5 were identified by selecting for functional properties. Eleven such promoters belonging to all three expression classes of the phage were analyzed. Their average AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. Interestingly, the consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for Escherichia coli RNA polymerase and can be used for the efficient in vitro synthesis of defined RNA species. In addition, some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eucaryotic translation systems.