Distinct functions of regions 1.1 and 1.2 of RNA polymerase σ subunits from Escherichia coli and Thermus aquaticus in transcription initiation

RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ(70) and T. aquaticus σ(A) subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ(70) subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the -10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation.     

Stabilizing interactions in the dimer interface of alpha-subunit in Escherichia coli RNA polymerase: a graph spectral and point mutation study

The formation of alpha(2) dimer in Escherichia coli core RNA polymerase (RNAP) is thought to be the first step toward the assembly of the functional enzyme. A large number of evidences indicate that the alpha-subunit dimerizes through its N-terminal domain (NTD). The crystal structures of the alpha-subunit NTD and that of a homologous Thermus aquaticus core RNAP are known. To identify the stabilizing interactions in the dimer interface of the alpha-NTD of E. coli RNAP, we identified side-chain clusters by using the crystal structure coordinates of E. coli alpha-NTD. A graph spectral algorithm was used to identify side-chain clusters. This algorithm considers the global nonbonded side-chain interactions of the residues for the clustering procedure and is unique in identifying residues that make the largest number of interactions among the residues that form clusters in a very quantitative way. By using this algorithm, a nine-residue cluster consisting of polar and hydrophobic residues was identified in the subunit interface adjacent to the hydrophobic core. The residues forming the cluster are relatively rigid regions of the interface, as measured by the thermal factors of the residues. Most of the cluster residues in the E. coli enzyme were topologically and sequentially conserved in the T. aquaticus RNAP crystal structure. Residues 35F and 46I were predicted to be important in the stability of the alpha-dimer interface, with 35F forming the center of the cluster. The predictions were tested by isolating single-point mutants alpha-F35A and alpha-I46S on the dimer interface, which were found to disrupt dimerization. Thus, the identified cluster at the edge of the dimer interface seems to be a vital component in stabilizing the alpha-NTD.     

Analysis of the spc ribosomal protein operon of Thermus aquaticus

The gene region of Thermus aquaticus corresponding to the distal portion of the S10 operon and to the 5'-portion of the Escherichia coli spc operon was cloned, using the E. coli gene for the ribosomal protein L5 as hybridization probe. The gene arrangement was found to be identical to E. coli, i.e. S17, L14, L24, L5, S14, S8 and L6. Stop and start regions of contiguous cistrons overlap, except for the S14-S8 intergenic region, whose size (67 bases) even exceeds the corresponding spacer regions in E. coli and Bacillus subtilis. A G + C content of 94% in third positions of codons was found in the ribosomal protein genes of T. aquaticus analyzed here. The stop codon of gene S17 (the last gene of the S10 operon in E. coli) and the start codon of gene L14 (the first gene of the spc operon in E. coli) overlap in T. aquaticus, thus leaving no space to accommodate an intergenic promoter preceding spc-operon-encoded genes in T. aquaticus. A possible promoter, localized within the S17 coding region, yielded only weak resistance (20 micrograms/ml) to chloramphenicol in E. coli and therefore could be largely excluded as the main promoter for spc-operon-encoded genes. We failed to detect a structure resembling the protein S8 translational repressor site, located at the beginning of the L5 gene in E. coli, in the corresponding region or any other region in the cloned T. aquaticus spc DNA.     

Inter- and intrasubunit interactions during the formation of RNA polymerase assembly intermediate

We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP) alpha and beta subunits during the formation of alpha(2)beta, a physiological RNAP assembly intermediate. We show that a 430-amino acid-long fragment containing beta conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with alpha. The alpha-interacting domain is held together by protein-protein interactions between beta segments F and I. Residues in catalytically important beta segments H and I directly participate in alpha binding; substitutions of strictly conserved segment H Asp(1084) and segment I Gly(1215) abolish alpha(2)beta formation in vitro and are lethal in vivo. The importance of these beta amino acids in alpha binding is fully supported by the structural model of the Thermus aquaticus RNAP core enzyme. We also demonstrate that determinants of RNAP assembly are conserved, and that a homologue of beta Asp(1084) in A135, the beta-like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest alpha-like subunit. However, the A135-AC40 interaction is weak compared with the E. coli alpha-beta interaction, and A135 mutation that abolishes the interaction is phenotypically silent. The results suggest that in eukaryotes additional RNAP subunits orchestrate the enzyme assembly by stabilizing weak, but specific interactions of core subunits.     

Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose

The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.