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.     

Structure and function of lineage-specific sequence insertions in the bacterial RNA polymerase beta' subunit

The large beta and beta' subunits of the bacterial core RNA polymerase (RNAP) are highly conserved throughout evolution. Nevertheless, large sequence insertions in beta and beta' characterize specific evolutionary lineages of bacteria. The Thermus aquaticus RNAP beta' subunit contains a 283 residue insert between conserved regions A and B that is found in only four bacterial species. The Escherichia coli RNAP beta' subunit contains a 188 residue insert in the middle of conserved region G that is found in a wide range of bacterial species. Here, we present structural studies of these two beta' insertions. We show that the inserts comprise repeats of a previously characterized fold, the sandwich-barrel hybrid motif (as predicted from previous sequence analysis) and that the inserts serve significant roles in facilitating protein/protein and/or protein/nucleic acid interactions.     

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.     

Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.     

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.     

Differences in Contacts of RNA Polymerases from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Thermus aquaticus</Emphasis> with <Emphasis Type="Italic">lac</Emphasis>UV5 Promoter Are Determined by Core-Enzyme of RNA Polymerase

The interaction of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter was studied at various temperatures. Using DNA-protein cross-linking induced by formaldehyde, it was demonstrated that each RNA polymerase formed a unique pattern of contacts with DNA in the open promoter complex. In the case of E. coli RNA polymerase, beta and sigma subunits were involved into formation of cross-links with the promoter, whereas in the case of T. aquaticus RNA polymerase its beta subunit formed the cross-links with the promoter. A cross-linking pattern in promoter complexes of a hybrid holoenzyme comprised of the core-enzyme of E. coli and sigma subunit of T. aquaticus was similar to that of the E. coli holoenzyme. This suggests that DNA-protein contacts in the promoter complex are primarily determined by the core-enzyme of RNA polymerase. However, temperature-dependent behavior of contact formation is determined by the sigma subunit. Results of the present study indicate that the method of formaldehyde cross-linking can be employed for elucidation of differences in the structure of promoter complexes of RNA polymerases from various bacteria.     

Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus aquaticus YT-1 and the deduced amino acid sequence of the enzyme

The gene for L-lactate dehydrogenase (LDH) from Thermus aquaticus YT-1 was cloned in Escherichia coli, using the Thermus caldophilus LDH gene as a hybridization probe, and its complete nucleotide sequence was determined. The LDH gene comprised 930 base pairs, starting with a GTG initiation codon. Its sequence had high homology (85.8% identity) with the LDH gene of T. caldophilus. The G + C content of the T. aquaticus gene was 70.9%, higher than that of the chromosomal DNA (67.4%). In particular, that in the third position of the codons used was 91.0%, similar to the T. caldophilus gene. The primary structure of T. aquaticus LDH was deduced from the nucleotide sequence of the LDH gene. It comprises 310 amino acid residues, as does T. caldophilus LDH, and its molecular mass was calculated to be 33,210 daltons. The amino acid sequence of the T. aquaticus LDH had 87.1% identity with that of the T. caldophilus LDH. At 23 positions, the respective residues differed in charge and polarity. These differences must be related to the differences in kinetic properties between the two enzymes. The constructed plasmid overproduced the T. aquaticus LDH in E. coli.     

An enhanced thermostability in thermophilic 5-S ribonucleic acids under physiological salt conditions.

The secondary structure of 5-S rRNAs of Thermus aquaticus (an extreme thermophile), Bacillus stearothermophilus (a moderate thermophile) and Escherichia coli (a mesophile) was compared using thermal denaturation techniques under varying ionic conditions. At a low ionic strength (10 mM K+), the Tm of T. aquaticus 5-S RNA differed by only 1 degrees C from that of E. coli RNA and the molecule was fully denatured well below the optimum growth temperature of the thermophile. The internal Na+, K+ and Mg2+ concentrations of T. aquaticus cells were determined to be 91 mM, 130 mM and 59 mM, respectively. Under these salt conditions, T. aquaticus 5-S RNA was significantly more stable than E. coli RNA and the 5-S RNA from B. stearothermophilus was intermediate as is its optimum growth temperature. The results suggest that the thermostability of macromolecules from thermophilic organisms may be specially dependent on the internal salt concentration. Furthermore, under these salt conditions, most of the secondary structure of the RNA remained stable at the optimum growth temperatures suggesting that ribosomal RNAs of thermophilic organisms contribute more to the thermostability of the ribosome than previously thought.