Michael J. Chamberlin

Physical and chemical characterization of two- and three-stranded adenine-thymine and adenine-uracil homopolymer complexes

Abstract The DNA homopolymers dA and dT have been prepared enzymically using Escherichia coli DNA polymerase, and their properties have been studied. Mixtures of dT § polymer with dA or rA polymers reacted rapidly to form the homopolymer pairs dA:dT and rA:dT. These complexes resembled the RNA pair rA:rU in being two-stranded helical structures containing complementary base pairs; however, the three homopolymer pairs differ in thermal stability. The homopolymer pair dA:rU is not stable under any conditions of temperature and salt concentration tested; mixtures of dA with rU contain three-stranded dA:rU+ 2 as the only helical complex. The other AT and AU homopolymer pairs can also form three-stranded complexes containing an additional equivalent of either dT or rU polymer under certain conditions.

Studies of the binding of Escherichia coli RNA polymerase to DNA: I. The role of sigma subunit in site selection☆

Abstract A membrane filter assay has been devised to study the binding of Escherichia coli RNA polymerase to DNA. RNA polymerase is quantitatively retained on nitrocellulose filters, and DNA bound in complexes between RNA polymerase and labeled T7 DNA is retained with an efficiency of up to 70%. The attachment of a single RNA polymerase molecule to a molecule of T7 DNA is sufficient to cause retention of the DNA molecule on the filter. Using the filter assay, it has been shown that both RNA polymerase holoenzyme and core polymerase bind to T7 DNA. The interaction of RNA polymerase holoenzyme with T7 DNA leads to two classes of complex. Approximately eight polymerase molecules can bind to each T7 DNA molecule to form a highly stable complex (Kassoc. = 1012 to 1014 m−1). In addition, there are many other sites on the DNA molecule at which a weak complex can be formed (Kassoc. = 108 to 108 m−1). The majority of holoenzyme molecules bound in the highly stable complex are thought to be bound at or near the T7 early promoter region, while the weak binding sites are thought to reflect a low, general affinity of holoenzyme for other regions of the DNA helix. In contrast, only a single major class of binding sites is found on T7 DNA for core polymerase (Kassoc. = 2 × 1011 m−1) and there are many such sites on the T7 genome. These sites are interpreted as reflecting a high general affinity of core polymerase for all regions of the DNA helix. It is concluded that RNA polymerase can exist in two conformational states, one programmed for promoter binding and initiation (holoenzyme) and the other designed for RNA chain elongation (core polymerase). Sigma subunit may function in site selection primarily to switch the enzyme reversibly between these two states.

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

Abstract A method for the isolation of homogeneous Escherichia coli RNA polymerase holoenzyme is described. The procedure involves the chromatography of a partially purified fraction of RNA polymerase containing both RNA polymerase holoenzyme and core polymerase on a phosphocellulose column in the presence of a high concentration of glycerol. Under these conditions, only small amounts of σ subunit are lost and RNA polymerase holoenzyme is well separated from the core polymerase.

Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli K12.

A given dose of ultraviolet irradiation produces as many pyrimidine-dimer-containing photoproducts in the DNA of the Escherichia coli Rec − strain JC1569 as it does in the related Rec + strain JC1557. Both strains excise dimer-containing photoproducts from their DNA to the same extent. In comparison with JC1557, approximately 30 times as much DNA of the Rec − strain is degraded per unit dose of ultraviolet light. Perhaps as a result of this excess ultraviolet-induced degradation, irradiated Rec − cells do not incorporate appreciable amounts of exogenous thymidine or thymine into DNA and have few survivors following exposure to an ultraviolet dose which almost all Rec + cells survive. A numerical argument is offered to establish the ability of Rec − cells successfully to replace excised pyrimidine-dimer-containing regions of the DNA with normal nucleotide residues. No effect of the rec − mutation in JC1569 was found on the level of activity of the following enzymes: endonuclease I, exonuclease I, exonuclease III and DNA phosphatase, and DNA polymerase and exonuclease II.

Physical and chemical characterization of the ordered complexes formed between polyinosinic acid, polycytidylic acid and their deoxyribo-analogues

This paper describes studies on the physical and chemical properties of the helical structures obtained when polydeoxycytidylic acid (dC) or its polyribo-analogue (rC) interacts with polydeoxyinosinic acid (dI) or its polyribo-analogue (rI). At moderate concentrations of a monovalent cation (less than 0·5 M ) the four complexes dI :dC, dI :rC, rI:dC and rI :rC are formed. These appear to be bihelical structures with perfect base pairing between the hypoxanthine residues of one strand and the cytosine residues of the other. The hybrid structures differ markedly from one another and from the dI:dC and rI:rC helices with respect to several properties.

Isolation and properties of transcribing ternary complexes of Escherichia coli RNA polymerase positioned at a single template base

We have studied the conditions needed for the formation of stable ternary complexes by Escherichia coli RNA polymerase using a procedure in which elongation by the majority of active enzyme molecules is halted at a specific template base. Stable complexes of this sort, containing RNA chains as short as 15 nucleotides, have been formed from three different promoter sites (T7 A1, lambda PL, and E. coli rrnB P1) using di- and trinucleotides as primers in reactions limited by the presence of only three of the nucleoside triphosphate substrates. The resulting ternary complexes can be stored for at least five days without loss in activity, and provide useful reagents and substrates for studies of the properties of RNA polymerases engaged in chain elongation and termination. At all three promoter sites abortive initiation, leading to synthesis and release of oligomers up to ten nucleotides, competes with productive initiation, leading to the formation of stable elongating complexes. Thus the relative instability of ternary RNA polymerase complexes bearing transcripts shorter than ten nucleotides may be a general feature of the transcription initiation reaction.

Mapping and characterization of transcriptional pause sites in the early genetic region of bacteriophage T7

During transcription of DNA templates in vitro, Escherichia coli RNA polymerase pauses at certain sequences before resuming elongation. Previous studies have established that some pausing events are brought about by the formation of RNA hairpin structures in the nascent transcript; however, it is not known whether this is an invariant and causal relationship. We have mapped and characterized almost 200 distinct pause sites located within the early region of bacteriophage T7 DNA using a collection of T7 deletion mutant DNAs and taking advantage of a procedure that permits synchronous transcription from the T7 A1 promoter. The pausing pattern is sensitive both to the overall concentration of nucleotide substrates and to the relative concentrations of the four nucleotides. The apparent Ks value for a particular nucleoside triphosphate can vary over a 500-fold range depending on the nucleotide sequence, and pausing at some sites can be induced by modest reductions in substrate concentrations. However, pausing is not solely a consequence of substrate limitation. Pausing at certain sites is caused by some feature of the template or of the transcript itself. Substitution of inosine triphosphate (ITP) for GTP during transcription strongly affects the pattern and strength of pausing events, suggesting that base-pairing interactions involving the RNA strand are important for some pausing events. Other pauses are determined by sequences downstream from the elongation site that have not yet been transcribed, and pausing at these sites is generally insensitive to substitution of IMP for GMP in the nascent transcript. Pausing at one particular site on T7 DNA is strongly enhanced by the presence of E. coli gene nusA protein. These results confirm that there are multiple classes of sites that lead to transcriptional pausing, and provide a collection of sites for further study. Using selected pause sites in the early region of T7 DNA, we have tried to evaluate the possible roles of primary sequence, base composition and secondary structure in pausing. Computer analysis was used to compare primary sequences and potential RNA hairpin structures in transcripts for pauses known to share similar biochemical properties. We see no correlation of pause sites with regions of particular base composition or with specific primary sequences. While some pauses are correlated with the potential to form stable RNA hairpins just upstream from the growing point of the RNA chain, there is not a strict one-to-one relationship between predicted RNA hairpins and the location of pause sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental and genetic regulation of bacillus subtilis genes transcribed by σ28-RNA polymerase

Sigma-28 RNA polymerase is a minor form of Bacillus subtilis RNA polymerase that is highly specific for transcription from a small number of promoter sites in the B. subtilis genome. We have followed transcription from two of these loci (P28-1 and P28-2) in vivo using a quantitative S1 nuclease mapping procedure. Both promoters are used at a modest rate in vegetatively growing cells (about 10 RNA copies per cell) and transcripts are initiated at the same start sites as found in vitro with the purified sigma 28-RNA polymerase. Transcription from the sigma 28 promoters varies somewhat with growth conditions and is shut off rapidly and almost completely after the first hour of sporulation. Neither sigma 28 transcripts is detected in vegetative cells of certain B. subtilis mutants (spoO classes A, B, E, and F) that are defective in sporulation. Transcription from these promoters is restored in second site revertants that are able to sporulate. Hence the action of sigma 28-RNA polymerase appears to be regulated by the spoO genes and the functions controlled by sigma 28-promoters may be closely tied to the system involved in the initiation of sporulation.

Studies of the binding of Escherichia coli RNA polymerase to DNA: IV. The effect of rifampicin on binding and on RNA chain initiation

Abstract Binding of Escherichia coli RNA polymerase to T7 DNA is not prevented by complexing of the enzyme with rifampicin, nor does rifampicin increase the rate of dissociation of the highly stable RNA polymerase holoenzyme-T7 DNA complex. Both free RNA polymerase and RNA polymerase-T7 DNA complexes are attacked by rifampicin in second-order reactions although the rate constant for the latter reaction is reduced about 100-fold. Alteration of the stability of the holoenzyme-DNA complex does not appreciably change the rate of rifampicin attack, nor does removal of sigma subunit from the RNA polymerase-DNA complex. It is suggested that the relative resistance of RNA polymerase holoenzyme-T7 DNA complexes to attack by rifampicin, when the drug is added together with the nucleoside triphosphates, is due to the rapid rate of RNA chain initiation by this complex.

Isolation of sigma-28-specific promoters from Bacillus subtilis DNA

Sigma-28-RNA polymerase is a minor form of RNA polymerase found in vegetative cells of Bacillus subtilis which utilizes promoter sites distinct from those recognized by the major RNA polymerase. We have isolated a collection of cloned B. subtilis DNA segments that contain in vitro promoter sites for sigma 28-RNA polymerase by screening a bacteriophage lambda library of B. subtilis genomic fragments. At least nine new sigma 28-specific promoter sites have been identified in this collection, and four have been partially mapped for further study. Our strategy employed a mix of RNA probes prepared by in vitro transcription with sigma 28-RNA polymerase of total B. subtilis DNA EcoRI and HindIII fragments. Over 70% of the unique clones identified contain sigma 28-specific promoter sites, suggesting that the method may have general application for identification of promoter-containing sequences. The efficiency with which sigma 28-specific promoters are detected is consistent with there being a relatively small number of such sites in the B. subtilis genome of which twelve have been cloned.