Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex
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Escherichia coli phage Qbeta RNA replicase, an RNA-dependent RNA polymerase (RNA-dependent RNA nucleotidyltransferase), is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors. Using a previously developed reconstitution system we have examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase activity. Renaturation is significantly more efficient when exogenously added native EF-Tu-Ts is crosslinked than when it is not. Crosslinked EF-Tu-Ts can be purified from a crude crosslinked postribosomal supernatant by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured Qbeta replicase. A sample of Qbeta replicase with crosslinked EF-Tu-Ts replacing the individual elongation factors was prepared. Although it lacked EF-Tu and EF-Ts activities, it could initiate transcription of both poly(C) and Qbeta RNA normally and had approximately the same specific activity as control enzyme. Denatured Qbeta replicase formed with crosslinked EF-Tu-Ts was found to renature much more rapidly than untreated enzyme and, in contrast to normal replicase, its renaturation was not inhibited by GDP. The results demonstrate that EF-Tu and EF-Ts function as complex in Qbeta replicase and do not perform their known protein biosynthetic function in the RNA synthetic reaction.
Original language | English |
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Journal | Proceedings of the National Academy of Science of the United States of America |
Volume | 73 |
Issue number | 4 |
Pages (from-to) | 1131-1135 |
Number of pages | 5 |
ISSN | 0027-8424 |
DOIs | |
Publication status | Published - 1976 |
Externally published | Yes |
Bibliographical note
Keywords: Chemical Phenomena; Chemistry; Coliphages; Peptide Elongation Factors; Protein Conformation; Q beta Replicase; RNA Nucleotidyltransferases; RNA, Viral; Structure-Activity Relationship
ID: 9298287