Different Roles for Basic and Aromatic Amino Acids in Conserved Region 2 of Escherichia coli ς70 in the Nucleation and Maintenance of the Single-stranded DNA Bubble in Open RNA Polymerase-Promoter Complexes

Abstract

Amino acid residues in region 2 of 70 have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with singlestranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the ؊10 region. We concluded that two basic amino acids (Lys 414 and Lys 418 ) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr 430 and Trp 433 ). It is likely that these four amino acids, which are close to each other in the structure of 70 , together are involved in the nucleation of the strand separation process.RNA synthesis in prokaryotes is carried out by a multisubunit RNA polymerase commonly referred to as the core enzyme (E).1 For promoter recognition, a sigma (initiation) factor is required; it interacts with the core polymerase to yield the holoenzyme (E), which is able to form an initiation-competent complex at promoter sequences in a multistep process involving conformational changes in both the protein and the DNA (1-3). A striking feature of such a complex is a region of strand separation that spans about 14 base pairs from the upstream edge of the conserved Ϫ10 promoter element to just beyond the start site of transcription initiation (4). It is thought that, kinetically, strand separation initiates in the Ϫ10 region and proceeds in a downstream direction. Measurement of the size and location of the region of strand separation as a function of temperature shows that at low temperatures a small single-stranded region can be detected that, as the temperature is increased, expands toward the start site (3, 5-7). In addition, the introduction of nicks and mismatches in the Ϫ10 region is more effective in the acceleration of open complex formation than if such distortions are introduced at a more downstream position (8, 9).The predominant sigma factor in Escherichia coli, which enables recognition of promoters of housekeeping genes, is referred to as 70 . Sequence comparison has shown that a large group of sigma factors shows significant homology to 70 . Four regions of sequence conservation have been identified, of which some have been subdivided to reflect the most extensive sequence conservation (10). A large body of data has implicated region 2.3 of the main sigma factors of E. coli, Bacillus subtilis, and other prokaryotes in the nucleation of the strand separation. This process eventually results in the formation of the active or open complex, possibly by facilitating base flipping of the highly conserved A at Ϫ11 of the nontemplate strand. The support...

Interaction of RNA polymerase with forked DNA: Evidence for two kinetically significant intermediates on the pathway to the final complex

“…Substitutions of alanine for aromatic amino acids in region 2.3 of 70 were shown to adversely affect formation of open complexes at promoters (12,22). Consistent with these observations, we show that the Y430A and W433A substitutions both reduce K f by 2.5-fold and K 1 to a lesser extent ( Table 1, compare line 1 with lines 5 and 6).…”

“…These values should be considered approximate because of the small changes in extents of binding detected for the slow phase over the duration of our observations (30 min). All the same, comparison of the ratios of rate constants indicates that the RF 3 formed with wt RNAP is 3-fold more stable than that containing YYW RNAP, which is qualitatively consistent with the results presented in Table 1 and elsewhere (12,22).…”

“…70 purification was carried out exactly as described (12,22). The substitutions in conserved region 2.3 of 70 have been described: the YW variant contains the Y430A and W433A substitutions; YYW and FYW additionally contain the Y425A or F427A substitutions, respectively (12,22).…”

“…The dissociation curves, reworked from experiments discussed in ref. 22, are biphasic. The fast phase (too fast for the manual mixing technique used here) corresponds to the dissociation of the heparin-sensitive complexes after the addition of heparin.…”

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

“…To elucidate the molecular procedures of base unpairing during open complex formation, altered forms of DNA (depurinated, nicked, forked, or with single-stranded gap) in the Ϫ10 region were used as templates (10,(14)(15)(16)(17)(18)(19). These studies suggested that base unpairing involves an initial distortion or localized melting event around Ϫ11 (16,20). In this paper, we provide results by using intact duplex DNA to demonstrate that the adenine residue itself at the position Ϫ11 of the nontemplate strand plays a master role in the initiation of base unpairing.…”