Characterization of the Low pH Solution Structure and Dynamics of the Region 4 ofEscherichia coliRNA Polymerase σ⁷⁰Subunit

Abstract

Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and [(1)H]-(15)N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of sigma(70)(4) protein in its protonated state.

Backbone dynamics of TFE‐induced native‐like fold of region 4 of Escherichia coli RNA polymerase σ70 subunit

“…A preliminary study allowed us to conclude that protonated rECσ in aqueous solution retains some residual α‐helical structure despite being almost unfolded 18. This conclusion was strongly supported by the changes observed in CD spectra of the protein upon stepwise addition of TFE 18.…”

“…The sequence of the loop fragment of ECσ exhibits almost no homology with the corresponding fragments in the domains of known structure, so the spatial organization of the HLHTH motif in the studied protein could not be precisely determined via homology modeling. That the sequence divergence in the loop region is reflected in substantial structural differences supported by our previous studies of the low‐pH solution structure of rECσ,18 which showed that the location of the loop in the HLHTH region differs by at least one residue from that found in the homologous proteins. In this context it should be recalled that ECσ assumes a noticeably different fold in complexes with replication factor RSD16 and T4‐phage AsiA factor 8.…”

“…We have studied by CD and NMR methods conformational propensity of a recombinant His 6 ‐tagged ECσ fragment in aqueous solution at low‐pH, at which conditions it is sufficiently soluble 17. Subsequent extensive CD and NMR studies on conformation and dynamics of the protonated rECσ,18 have demonstrated that it is mostly unfolded, as was also predicted by sequence analysis alone,19 but three regions tend to assume a helical structure with a propensities to adopt a fold similar to that of the canonical HLHTH DNA‐binding motif. The spatial organization of this motif has been determined for structures of homologous σ 4 domains of T. aquaticus ,14 T. thermophilus ,15 and T. maritima ,8 and of a distant one, σ from Aquifex aeolicus (Ref.…”

“…Preparation of recombinant 107‐residue fragment of ECσ, rECσ, containing C‐terminal residues 528–613 of σ 70 comprising a C‐terminal fragment of region 3.2 and the whole domain 4 preceded by a 21‐amino acid segment MGSSHHHHHHSSGLVPRGSHM carrying (His) 6 ‐Tag followed by thrombin cleavage site, was described previously 18. Resonance assignmentswere done for 13 C and 15 N uniformly double‐labeled rECσ,28 whereas 15 N relaxation was measured for 15 N labeled protein.…”

“…Owing to the specific solvation of peptide groups24, 25 TFE induces closure of individual intramolecular hydrogen bonds26 and thus may propagate context‐dependent formation of native‐like secondary structures in polypeptides 27. Using CD spectroscopy we have shown that TFE indeed induces the buildup of helical structures,18 the population of which increases significantly upon stepwise addition of TFE. Furthermore, TFE content even as low as 3% (v/v) prevented protein aggregation accompanying deprotonation of Glu and Asp side chain carboxyl groups, which enabled us to study the protein structural properties at pH 4.5, which was closer to the physiological pH value than was the acidic pH required for ECσ to remain soluble in pure aqueous solution 17.…”