Mapping of the priming substrate contacts in the active center of Escherichia coli RNA polymerase.

Mapping of Subunit−Subunit Contact Surfaces on the β Subunit of Escherichia coli RNA Polymerase

“…Location of R Subunit Contact Site(s) on the β Subunit. Taken all of our observations together and as shown in Figure 6, we propose that (i) the primary contact site of R subunit on the β subunit is located at the central portion between residues N737 and R936 including the conserved regions F and G; and (ii) the extreme C-terminal region between residues A1139 and G1318, including the conserved region I and the putative catalytic center for RNA polymerization (30,31), plays a regulatory role in the binary complex formation. The intermediate region between these two domains for R assembly may not be involved in this process because at least parts of this region (the dispensable region II) can be deleted without affecting the assembly and function of RNA polymerase (29).…”

“…The protease-sensitive central part of the β subunit between residues R540/R542 and R801 includes the conserved regions D and E. It should be worthwhile to note that the binding sites for rifampicin (32,33), streptolydigin (34), priming substrates (30,35), and the stringent factor ppGpp (36,37) are all clustered in this protease-sensitive and supposedly surface-exposed region. Furthermore, one of the two contact sites of the σ 70 subunit on β is also located in this region (38).…”

HO.bul. and DNase I Probing of E.sigma.70 RNA Polymerase-.lambda.PR Promoter Open Complexes: Mg2+ Binding and Its Structural Consequences at the Transcription Start Site

“…A similar pocket on Ea70 may surround the -4 to +1 nucleotides on the t strand. In addition to the carboxylate residues implicated in nucleotide (and possibly Mg2+) binding, histidine 1237 and lysine 1065 (Mustaev et al, 1991) and lysine 1234 or 1242 (Grachev et al, 1989) of fi and region 3 of a70 (Severinov et al, 1994) are reported to be part of the catalytic center and may also lie in the catalytic pocket. In a model where the conversion from RP"i to RPo2 results from charge effects alone, local negative charge density due to the Ea70 carboxylates and DNA phosphates may be localized in this pocket in RP0|, excluding Fe(EDTA)2' and Mn04", Because they react with Mn04", n strand thymine bases at -4 and -3 (and perhaps +2) are evidently outside of the region of high local negative charge density.…”

A structure/function analysis ofEscherichia coliRNA polymerase

“…A second major impetus for detailed study of |3 was the Ending that three distinct regions in (3 are in close proximity to the 5 ' end of the nascent transcript. Crosslinking indicates that lysl 065 in conserved region H, his 123 7 in conserved region I (Mustaev al. 1991) and a residue in between amino acids 516 and 540 (Cluster 1 of the Rif region; see figure 3) are close to the 5' end of the nascent RNA transcript (Severinov et al 19956).…”

“…1991) and a residue in between amino acids 516 and 540 (Cluster 1 of the Rif region; see figure 3) are close to the 5' end of the nascent RNA transcript (Severinov et al 19956). Crosslinking indicates that lysl065 in conserved region H, his 123 7 in conserved region I (Mustaev et al 1991) and a residue in conserved region D are close to the 5' end of the nascent RNA transcript (A. Goldfarb, personal communication). Amino acids in the second largest subunit of eukaryotic RNA polymerases that are homologous to lysl065 also crosslink to the 5' face of the nascent transcript, indicating that the relation between polymerase and transcript described in prokaryotes has been preserved throughout evolution.…”