TY - JOUR
T1 - Interactions of Escherichia coli transcription termination factor rho with RNA. II. Electron microscopy and nuclease protection experiments
AU - Bear, David G.
AU - Hicks, Pamela S.
AU - Escudero, Kenneth W.
AU - Andrews, Christopher L.
AU - McSwiggen, James A.
AU - von Hippel, Peter H.
N1 - Funding Information:
We gratefully acknowledge Dr Jack D. Griffith (and the Lineberger Cancer Research Center Electron Microscopy Facility at the University of North Carolina) for vital technical help in obtaining the initial electron microscope images of rho protein, and for continued advice and moral support throughout the project. In addition, we are pleased to acknowledge helpful discussions with Drs John Trotter, David Draper and Stephen Kowalczykowski, and to thank Drs John Trotter, Robert Kelley and Leonard Napolitano of t)he 1Jniversity of New Mexico School of Medicine for the development and organization of an excellent electron microscopy facility. This research was supported in part by NSF fellowships to J.A.M. and K.W.E., a predoctoral traineeship on USPHS Training grant GM-00715 to J.A.M., USPHS Postdoctoral Fellowship GM-06676 and USPHS Research grant GM-32055 to D.G.B., and USPHS grants GM-15792 and GM21958 to P.H.v.H. A preliminary account of a portion of this study has appeared in the 1986 Proceedings of the Annual Meeting of the Electron Microscopy Society of America.
PY - 1988/2/20
Y1 - 1988/2/20
N2 - Structural aspects of the interaction between Escherichia coli transcription termination factor rho and RNA have been investigated, using nuclease protection assays and electron microscopy. A synthetic RNA, poly(rC), has been used as a substrate for these studies, since it binds tightly to rho and acts as a strong activator of the ATPase activity of rho. Digestion of oligo(rC)-rho complexes with ribonuclease A yields oligo(rC) fragments with a maximum length of 70 to 80 nucleotide residues. Electron micrographs demonstrate that rho binds to poly(rC) as a toroid-shaped oligomer with an outside diameter of approximately 120 Å. Taken together with data from the accompanying paper, which shows that the RNA binding site size per rho monomer is 13(±1) nucleotide residues, we infer that rho binds RNA as a hexamer with an oligomeric site size of 72 to 84 residues. Further analysis of the electron micrographs has revealed that the polynucleotide chain is wrapped around, or condensed within, the protein oligomer, rho hexamers bind to poly(rC) with moderate co-operativity (ω = 380 ± 60), displaying no significant preference for binding to chain ends versus internal sites on the polynucleotide chain. These findings and those of the companion paper are discussed in terms of various models for the structure of the rho-RNA complex in transcription termination.
AB - Structural aspects of the interaction between Escherichia coli transcription termination factor rho and RNA have been investigated, using nuclease protection assays and electron microscopy. A synthetic RNA, poly(rC), has been used as a substrate for these studies, since it binds tightly to rho and acts as a strong activator of the ATPase activity of rho. Digestion of oligo(rC)-rho complexes with ribonuclease A yields oligo(rC) fragments with a maximum length of 70 to 80 nucleotide residues. Electron micrographs demonstrate that rho binds to poly(rC) as a toroid-shaped oligomer with an outside diameter of approximately 120 Å. Taken together with data from the accompanying paper, which shows that the RNA binding site size per rho monomer is 13(±1) nucleotide residues, we infer that rho binds RNA as a hexamer with an oligomeric site size of 72 to 84 residues. Further analysis of the electron micrographs has revealed that the polynucleotide chain is wrapped around, or condensed within, the protein oligomer, rho hexamers bind to poly(rC) with moderate co-operativity (ω = 380 ± 60), displaying no significant preference for binding to chain ends versus internal sites on the polynucleotide chain. These findings and those of the companion paper are discussed in terms of various models for the structure of the rho-RNA complex in transcription termination.
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U2 - 10.1016/0022-2836(88)90306-3
DO - 10.1016/0022-2836(88)90306-3
M3 - Article
C2 - 2451029
AN - SCOPUS:0023898207
SN - 0022-2836
VL - 199
SP - 623
EP - 635
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -