Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex

Stanley Brown; Thomas Blumenthal

doi:10.1073/pnas.73.4.1131

Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex

Stanley Brown, Thomas Blumenthal

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

21 Citationer (Scopus)

Abstract

Udgivelsesdato: 1976-Apr

Originalsprog	Engelsk
Tidsskrift	Proceedings of the National Academy of Science of the United States of America
Vol/bind	73
Udgave nummer	4
Sider (fra-til)	1131-1135
Antal sider	5
ISSN	0027-8424
DOI	https://doi.org/10.1073/pnas.73.4.1131
Status	Udgivet - 1976
Udgivet eksternt	Ja

Bibliografisk note

Keywords: Chemical Phenomena; Chemistry; Coliphages; Peptide Elongation Factors; Protein Conformation; Q beta Replicase; RNA Nucleotidyltransferases; RNA, Viral; Structure-Activity Relationship

Adgang til dokumentet

10.1073/pnas.73.4.1131

Citationsformater

@article{b896ff50d29011dd9473000ea68e967b,

title = "Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex",

abstract = "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.",

author = "Stanley Brown and Thomas Blumenthal",

note = "Keywords: Chemical Phenomena; Chemistry; Coliphages; Peptide Elongation Factors; Protein Conformation; Q beta Replicase; RNA Nucleotidyltransferases; RNA, Viral; Structure-Activity Relationship",

year = "1976",

doi = "10.1073/pnas.73.4.1131",

language = "English",

volume = "73",

pages = "1131--1135",

journal = "Proceedings of the National Academy of Science of the United States of America",

issn = "0027-8424",

publisher = "The National Academy of Sciences of the United States of America",

number = "4",

}

TY - JOUR

T1 - Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex

AU - Brown, Stanley

AU - Blumenthal, Thomas

N1 - Keywords: Chemical Phenomena; Chemistry; Coliphages; Peptide Elongation Factors; Protein Conformation; Q beta Replicase; RNA Nucleotidyltransferases; RNA, Viral; Structure-Activity Relationship

PY - 1976

Y1 - 1976

N2 - 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.

AB - 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.

U2 - 10.1073/pnas.73.4.1131

DO - 10.1073/pnas.73.4.1131

M3 - Journal article

C2 - 1063392

SN - 0027-8424

VL - 73

SP - 1131

EP - 1135

JO - Proceedings of the National Academy of Science of the United States of America

JF - Proceedings of the National Academy of Science of the United States of America

IS - 4

ER -