Complex of transfer-messenger RNA and elongation factor Tu. Unexpected modes of interaction.
Transfer-messenger RNA (tmRNA) is a stable RNA in bacteria of 360 +/- 40 nucleotides that can be charged with alanine and can function as both tRNA and mRNA. Ribosomes that are stalled either in a coding region of mRNA or at the 3' end of an mRNA fragment lacking a stop codon are rescued by replacing their mRNA for tmRNA. Here we demonstrate that the interaction of tmRNA with the elongation factor Tu shows unexpected features. Deacylated tmRNA can form a complex with either EF-Tu.GDP or EF-Tu.GTP, the association constants are about one order of magnitude smaller than that of an Ala-tRNA.EF-Tu.GTP complex. tmRNA as well as Ala-tmRNA can be efficiently cross-linked with EF-Tu.GDP using a zero-length cross-link. The efficiency of cross-linking in the case of deacylated tmRNA does not depend on an intact CCA-3' end and is about the same, regardless whether protein mixtures such as the post-ribosomal supernatant (S100 enzymes) or purified EF-Tu are present. Two cross-linking sites with EF-Tu.GDP have been identified that are located outside the tRNA part of tmRNA, indicating an unusual interaction of tmRNA with EF-Tu.GDP.
Zvereva
M I
MI
Department of Chemistry, Moscow State University, Moscow 119899, Russia.
Ivanov
P V
PV
Teraoka
Y
Y
Topilina
N I
NI
Dontsova
O A
OA
Bogdanov
A A
AA
Kalkum
M
M
Nierhaus
K H
KH
Shpanchenko
O V
OV
D002474:Cell-Free System; D018388:Codon, Terminator; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000494:pharmacology
D018388:Codon, Terminator; D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D006153:Guanosine Diphosphate; D012313:RNA; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D014357:Trypsin; D010444:Peptide Elongation Factor Tu