Inter-subunit recognition and manifestation of segmental mobility in Escherichia coli RNA polymerase: a case study with omega-beta' interaction.
Omega (omega), consisting of 91 amino acids, is the smallest of all the Escherichia coli RNA polymerase subunits and is organized into an N-terminal domain of 53 amino acids followed by an unstructured tail in the C-terminal region. Our earlier experiments have shown a chaperone-like function of omega in which it helps to maintain beta' in a correct conformation and recruit it to the alpha(2)beta subassembly to form a functional core enzyme (alpha(2)betabeta'omega). The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch onto the N-terminal and C-terminal ends of the beta'-subunit. In the present study we have monitored the conformational changes in beta' as the denatured protein is refolded in the presence and absence of omega using tryptophan fluorescence emission of beta' as well as acrylamide quenching of Trp fluorescence. Results indicate that the presence of stoichiometric amounts of omega is helpful in beta' refolding. We have also monitored the behavior of the C-terminal tail of omega by engineering three cysteine residues at three different sites in omega and subsequently labeling them with a sulphydryl-specific fluorescent probe. Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of omega is mobile in the free protein and gets restrained in the presence of beta'. Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the beta' subunit whereas the end of the C-terminal of omega, although it is restrained in the presence of beta', has no interacting partner within a 4-A radius.
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, A.P., India.
Pallavi
P
Ghosh
Chandrashekharan
C
Ramakrishnan
Dipankar
D
Chatterji
D020106:Acrylamide / Q000737:chemistry; D000595:Amino Acid Sequence; D003545:Cysteine / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D005454:Fluorescence Polarization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013824:Thermus / Q000201:enzymology / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry
D009282:Naphthalenesulfonates; D021122:Protein Subunits; D020106:Acrylamide; D014364:Tryptophan; D012321:DNA-Directed RNA Polymerases; D003545:Cysteine