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M J Chamberlin

Publications and source records attributed to M J Chamberlin.

14 recordsLinked to original sources
PubMed1979

Utilization of promoter and terminator sites on bacteriophage T7 DNA by RNA polymerases from a variety of bacterial orders.

The transcriptional properties of bacterial RNA polymerases purified from seven different species and representing a variety of bacterial orders have been studied using the well characterized DNA from phage T7 as template. The subunit composition of the different preparations suggests that each RNA polymerase holoenzyme has a promoter structure (betabeta'alpha2sigma) similar to that of the well studied E. coli and B. subtilis enzymes. Each enzyme utilizes DNA from bacteriophage T7 as an effective template for RNA synthesis, although all preparations contain a substantial fraction of inactive enzyme. Electrophoretic analysis of the RNA products made with the different RNA polymerases in vitro using T7 DNA (deletion mutant deltaD111) as template reveals that with minor exceptions, all of the heterologous RNA polymerases utilize the same collection of promoter sites on T7 used by the E. coli host enzyme, and only those promoter sites. The T7 early terminator is also efficiently utilized by each enzyme. Since the different bacterial species from which the RNA polymerases were derived are genetically quite distant, it appears that there is a structural element in the promoter which governs its recognition and which is universally recognized among RNA polymerases of different bacterial species. While the different bacterial RNA polymerases generally utilize the same set of T7 promoter sites, the efficiency of utilization of the different promoters varies considerably for different RNA polymerases and for different reaction conditions with the same RNA polymerase. Hence although the several T7 promoter sites share the ability to be recognized by bacterial RNA polymerases, each shows a unique pattern of utilization and therefore must possess a unique element of promoter structure as well. It has previously been shown (Stahl and Chamberlin, 1977) that T7 promoters A1, C, D and E interact differently with E. coli RNA polymerase as judged by the properties of complexes formed between each promoter and the latter enzyme. Since competition takes place among different promoter sites on a template and since these sites can differ functionally, small changes in reaction conditions or in the structure of the RNA polymerase can lead to significant changes in the rate of utilization of different promoter sites even when these promoter sites share common elements. Because the T7 promoters and terminator are utilized efficiently by such a wide range of RNA polymerases and because each of the several T7 promoters possesses unique properties which govern its utilization by RNA polymerase, analysis of the transcripts formed on a T7 DNA template provides a simple and rapid procedure for detecting and analyzing alterations in bacterial RNA polymerases which affect promoter or terminator recognition or utilization.

Bacteria↗
PubMed1979

Altered promoter selection by a novel form of Bacillus subtilis RNA polymerase.

Bacillus subtilis RNA polymerase holoenzyme prepared by several standard methods utilizes bacteriophage T7 DeltaD111 DNA as an efficient template. The major RNA products are specific transcripts from T7 promoters A(1) and C; these promoters are also efficiently utilized by RNA polymerases purified from a wide range of other bacterial species [Wiggs, J., Bush, J. & Chamberlin, M. (1979) Cell 16, 97-109]. In contrast, B. subtilis RNA polymerase preparations purified by a modification of the method of Burgess and Jendrisak (designated fraction 5) utilize T7 DeltaD111 promoters A(1) and C and an additional promoter site, J, which has been located at 90.6% on the standard T7 physical map. This promoter is not used by B. subtilis core RNA polymerase or by RNA polymerase from any other bacterial species we have tested. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of fraction 5 RNA polymerase shows that it contains B. subtilis components sigma and delta and a polypeptide of M(r) 92,000 in addition to the B. subtilis beta, beta', and alpha subunits. Chromatography of fraction 5 on single-stranded DNA-cellulose gives an enzyme fraction, Bs I, that is indistinguishable from B. subtilis RNA polymerase holoenzyme both in its peptide composition (betabeta'alpha(2)sigma) and in the selective transcription of only T7 RNAs A(1) and C. Chromatography of fraction 5 on phosphocellulose yields an enzyme fraction, Bs II, devoid of sigma subunit but containing the M(r) 92,000 peptide and traces of delta. This fraction synthesizes predominantly T7 J RNA in vitro together with traces of T7 A(1) and C RNAs. Hence, B. subtilis RNA polymerase fraction Bs II appears to contain a form of RNA polymerase that can transcribe selectively without detectable amounts of B. subtilis sigma subunit and that utilizes a promoter site not used by other known bacterial RNA polymerases. The structural basis for this specificity is not yet known.

Bacillus subtilis↗
PubMed1979

Mapping of class II promoter sites utilized in vitro by T7-specific RNA polymerase on bacteriophage T7 DNA.

Restriction endonuclease Bgl II cleaves T7 DNA at a unique site (28.76% on the standard T7 map), yielding two fragments of molecular weights 18.9 x 10(6) (A) and 7.6 x 10(6) (B). Fragment B, representing the leftmost portion of the genome, has been purified by zone sedimentation. Transcription of fragment B by T7-specific RNA polymerase gives only r-strand-specific RNA. Analysis of the products by polyacrylamide gel electrophoresis reveals four major RNA species which have apparent molecular weights of 2.1 x 10(6), 1.36 x 10(6), 0.85 x 10(6) and 0.125 x 10(6), respectively. Each of these RNAs is reduced in size when transcription is carried out with fragment B, which has been shortened by treatment with Escherichia coli exonuclease III. Therefore, each of the transcripts must be terminated at the right end of fragment B. Analysis of the molecular weights of the four transcripts produced from whole and from exonucleolytically shortened fragment B suggests that these transcripts are read from promoters located at 13.5, 18.9, 22.6, and 27.9%, respectively, on the standard T7 map. Hence, there are at least four promoters governing the transcription of the class II region. Transcripts initiated at these promoters on intact T7 DNA appear to read through the class II and part of the class III genetic region and terminate at the strong terminator for T7-specific RNA polymerase near 61%. Transcription of fragment B which has been cleaved with the restriction endonuclease Hpa I seems to activate a fifth promoter for T7-specific RNA polymerase. This promoter appears to be identical to the promoter previously described by Oakley and Coleman (Proc. Natl. Acad. Sci. U.S.A. 74:4266-4270, 1977) that maps near 15% on the standard T7 map. Little or no RNA is read from T7 Bgl II fragment B, which has a mobility expected for a transcript read from this promoter. However, upon cleavage with Hpa I, this promoter is utilized approximately 10-fold more efficiently than the other class II promoters. The mechanism of this activation is not yet known.

Coliphages↗
PubMed1978

Transcription of T7 DNA containing modified nucleotides by bacteriophage T7 specific RNA polymerase.

The interaction of bacteriophage T7 specific RNA polymerase with its cognate promoter sites has been probed by selectively replacing bases in one T7 promoter site with base analogs. Base analogs such as 2,6-diaminopurine or hypoxanthine, which alter residues appearing in the minor groove of the DNA helix, prevent utilization of the promoter by T7 RNA polymerase. These analogs do not affect transcription which starts outside of the modified region. In contrast, base analogs that have alterations that appear in the major groove of the DNA helix, such as uracil, 5-bromouracil, 5-methylcytosine, 5-hydroxymethylcytosine, and [5-HgSR]pyrimidines, do not prevent utilization of the promoter. The deoxyribonucleoside analog 5'-imino-5'-deoxythymidine, an alteration appearing in the deoxyribose-phosphodiester backbone of the DNA helix, does not prevent promoter recognition. Haemophilus aegyptius restriction endonuclease III, which cleaves DNA at the sequence 5'GGCC3', does not act at sites in which the guanine residues in one of the two DNA strands have been substituted with hypoxanthine. This implicates the guanine amino group in the minor groove of the DNA helix as a possible recognition point for this restriction endonuclease.

Base Sequence↗
PubMed1977

Binding of Escherichia coli RNA polymerase to T7 DNA. Displacement of holoenzyme from promoter complexes by heparin.

Escherichia coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase-T7 DNA complex formed at the major T7 A1 promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter A1. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter A1. Thus, the rate of inactivation of RNA polymerase-T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.

Binding Sites↗
PubMed1977

Electron microscope studies of transient complexes formed between Escherichia coli RNA polymerase holoenzyme and T7 DNA.

Electron microscopy was used to study the formation of random complexes between Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a promoterless fragment (Mbo I-C) of bacteriophage T7 DNA, and to determine the location of the polymerase molecules bound at 3 degrees to the promoter-containing (Hinf)1100 fragment of the same DNA. The value of the Ka of random binding is about 3 times 10(4)M-1 when the enzyme is slowly diluted from its storage condition and is incubated with DNA for up to 2 min at 37 degrees. If dilution is rapid and occurs in a single step, or if incubation extends beyond 5 min, a substantial portion of RNA polymerase is converted to a form that binds randomly with a much greater affinity (about 10(8)M-1). Hence true random binding by RNA polymerase holoenzyme is much weaker than previously thought. However, great caution is required in assessing the extent of random binding where damage to the enzyme may occur. When RNApolymerase holoenzyme is incubated at 0 degrees with promoter-containing fragment (Hinf)1100, complexes form at the same promoter sites utilized at 37 degrees, although the highly stable "open" promoter complex is not formed under these conditions. However, the extent of binding is reduced as compared to promoter complexes formed at 37 degrees. This gives direct evidence for formation of complexes with promoter sites that have properties of the hypothetical "closed"complexes formed between RNA polymerase and duplex DNA.

Coliphages↗
PubMed1977

T7- and T3-specific RNA polymerases: characterization and mapping of the in vitro transcripts read from T3 DNA.

The late genetic region of the T3 bacteriophage genome is transcribed by a phage-specified RNA polymerase, the product of T3 gene 1. In vitro, purified T3 RNA polymerase acting with T3 DNA template synthesizes six major RNA products, with molecular weights of 5.5 X 10(6), 4.2 X 10(6), 1.7 X 10(6), 0.87 X 10(6), 0.52 X 10(6), and 0.23 X 10(6). These are designated T3 RNA species I through VI, respectively. These RNAs are formed in equimolar amounts, with the exception of T3 species V, which is made in approximately twice this amount. T3 RNA species I, II, and VI have been mapped by the use of terminally deleted templates and are found to originate from promoters located at 56, 67, and 98% genome length, respectively, and to share a common terminator at approximately 100%. T3 species III, IV, and V must originate from the class III region of the T3 genome between 37.5 and 56%. Although the pattern of transcription by T3 RNA polymerase in vitro closely resembles that of the RNA polymerase from the related phage T7, neither polymerase is able to use major promoter sites for the other enzyme at an appreciable rate.

Coliphages↗
PubMed1977

Transcriptional termination at the end of the early region of bacteriophages T3 and T7 is not affected by polarity suppressors.

Bacterial mutations of known rho genotype (psu-1 through psu-4) were shown to have no effect on transcriptional termination at the termination site at the end of the early region of T3 and T7 DNAs. Transcriptional termination was assayed in these bacterial mutants by comparing the patterns of late protein production produced by T3 and T7 amber I bacteriophage infection. All the rho (psu) mutants tested showed the same pattern of late protein production as the wild-type strain (rho+ or psu+) after T3 or T7 amber I infection. The presence of the mutant rho allele during bacteriophage infection, therefore, did not allow the host RNA polymerase to read through the terminator located at approximately 20% of the T3 and T7 genomes. These results suggest that rho factor may not be involved in reading of the T3 and T7 20% terminators in vivo.

Coliphages↗