title: A new kinetic model reveals the synergistic effect of E-, P- and A-sites on +1 ribosomal frameshifting.
abstract: Programmed ribosomal frameshifting (PRF) is a process by which ribosomes produce two different polypeptides from the same mRNA. In this study, we propose three different kinetic models of +1 PRF, incorporating the effects of the ribosomal E-, P- and A-sites toward promoting efficient +1 frameshifting in Escherichia coli. Specifically, the timing of E-site tRNA dissociation is discussed within the context of the kinetic proofreading mechanism of aminoacylated tRNA (aa-tRNA) selection. Mathematical modeling using previously determined kinetic rate constants reveals that destabilization of deacylated tRNA in the E-site, rearrangement of peptidyl-tRNA in the P-site, and availability of cognate aa-tRNA corresponding to the A-site act synergistically to promote efficient +1 PRF. The effect of E-site codon:anticodon interactions on +1 PRF was also experimentally examined with a dual fluorescence reporter construct. The combination of predictive modeling and empirical testing allowed the rate constant for P-site tRNA slippage (k(s)) to be estimated as k(s) approximately 1.9 s(-1) for the release factor 2 (RF2) frameshifting sequence. These analyses suggest that P-site tRNA slippage is the driving force for +1 ribosomal frameshifting while the presence of a 'hungry codon' in the A-site and destabilization in the E-site further enhance +1 PRF in E. coli.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.
authors.forename: Pei-Yu
authors.initials: PY
authors.lastname: Liao
authors.forename: Prateek
authors.initials: P
authors.lastname: Gupta
authors.forename: Alexey N
authors.initials: AN
authors.lastname: Petrov
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Dinman
authors.forename: Kelvin H
authors.initials: KH
authors.lastname: Lee
mesh_terms: D000926:Anticodon / Q000737:chemistry; D001665:Binding Sites; D003062:Codon / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D018965:Frameshifting, Ribosomal; D007700:Kinetics; D008957:Models, Genetic; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000926:Anticodon; D003062:Codon; D012346:RNA, Transfer, Amino Acyl; D012343:RNA, Transfer