Interrupted catalysis: the EF4 (LepA) effect on back-translocation.
EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST-->I(1)-->I(2)-->I(3)-->PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3'-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I(3) rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I(3) is likely to be important for optimizing functional protein biosynthesis.
Liu
Hanqing
H
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
Pan
Dongli
D
Pech
Markus
M
Cooperman
Barry S
BS
D001483:Base Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D010448:Peptide Initiation Factors; D011691:Puromycin / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; C515651:LepA protein, E coli; D010448:Peptide Initiation Factors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D035602:Transcriptional Elongation Factors; C019037:tRNA, peptidyl-; D011691:Puromycin