Studies of the functional topography of Escherichia coli RNA polymerase. Affinity labelling of RNA polymerase in a promoter complex by phosphorylating derivatives of primer oligonucleotides

Amidation of the 5'-phosphate group of the heptanucleotide pdApdApdApdTpdCpdGprC and of its derivatives of the general formula (pdN)npdGprC (n = 0-5) with imidazole, N-methylimidazole, and 4-dimethylaminopyridine afforded a series of phosphorylating affinity reagents. The parent oligonucleotides of this series complementary to promoter A2 of T7 phage over the region (-5 to +2) are known to be efficient primers of the synthesis of RNA by Escherichia coli RNA polymerase with promoter A2 as template. Treatment of the complex RNA-polymerase X promoter-A2 with affinity reagents followed by addition of [alpha-32P]UTP resulted in labelling of RNA polymerase by the residues -(pdN)npdGprCprU (p = radioactive phosphate). This affinity labelling was highly selective because elongation of the covalently bound residues (pdN)npdGprC by prU residues was catalyzed by the active center of RNA polymerase. The most efficient reagents were N-methylimidazolides. A dramatic change of the pattern of labelling of the subunits beta, beta', and sigma took place with changing n. Maximum labelling of the beta subunit occurred at n = 1 and of the sigma subunit at n = 5. The targets in both the subunits were His residues. The alpha subunit was not specifically labelled.     

Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.     

Studies on the functional topography of Escherichia coli RNA polymerase. Highly selective affinity labelling by analogues of initiating substrates

RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [alpha-32P]NTP. Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNpN (where p = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive. Labelling took place only when the combination of the reagent and [alpha-32P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short 'arms', only the beta subunit was labelled; the targets were His and/or Lys residues. With reagents having longer 'arms', the sigma subunit was also labelled.     

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].     

Localization of lysine residues in the site of initiating substrate binding of E. coli RNA-polymerase

Superselective affinity labelling of E. coli RNA polymerase in a complex with the promoter-containing fragment of T7 DNA by treatment with orto-formylphenyl ester of GMP followed by addition of [alpha-33P]UTP resulted in covalent binding of the residue--pGpU (p-radioactive phosphate) with one of lysine residues of the beta-subunit, Lys1048, Lys1051, Lys1057, Lys1065. The amino acid sequence of this region of the beta-subunit of E. coli RNA polymerase has a high extent of homology with that deduced for a region of tobacco chloroplast RNA polymerase on the basis of the nucleotide sequence of the chloroplast rpoB-like gene.     

Highly selective affinity labeling of DNA-dependent RNA-polymerase of bacteriophage T7

T7 phage RNA polymerase was affinity labelled in the presence of its promoter by treatment with an ATP gamma-derivative (a phosphoamide obtained from 4-(N-chloroethyl, N-methyl)aminobenzylamine, or one of esters obtained from 2-methoxy-4-formylphenol, 4-formylphenol, and 2[N-(4-formylphenyl), N-methyl]-aminoethanol) followed by addition of [alpha-32P]GTP. The most efficient labelling took place with the alkylating phosphoamide reagent.     

Sequence and analysis of the gene for bacteriophage T3 RNA polymerase.

The RNA polymerases encoded by bacteriophages T3 and T7 have similar structures, but exhibit nearly exclusive template specificities. We have determined the nucleotide sequence of the region of T3 DNA that encodes the T3 RNA polymerase (the gene 1.0 region), and have compared this sequence with the corresponding region of T7 DNA. The predicted amino acid sequence of the T3 RNA polymerase exhibits very few changes when compared to the T7 enzyme (82% of the residues are identical). Significant differences appear to cluster in three distinct regions in the amino-terminal half of the protein. Analysis of the data from both enzymes suggests features that may be important for polymerase function. In particular, a region that differs between the T3 and T7 enzymes exhibits significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. The region that flanks the structural gene contains a number of regulatory elements including: a promoter for the E. coli RNA polymerase, a potential processing site for RNase III and a promoter for the T3 polymerase. The promoter for the T3 RNA polymerase is located only 12 base pairs distal to the stop codon for the structural gene.     

Mutational analysis of an extracytoplasmic-function sigma factor to investigate its interactions with RNA polymerase and DNA

The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.