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A new kinetic model reveals the synergistic effect of E-, P- and A-sites on +1 ribosomal frameshifting.

Liao PY, Gupta P, Petrov AN, Dinman JD, Lee KH - Nucleic Acids Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.

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.

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The three kinetic models for +1 PRF in E. coli. Steps I–VI illustrate the non-frameshifting translation elongation process: I. initial binding; II codon recognition; III. GTPase activation; IV. GTP hydrolysis; V. EF-Tu dissociation; VI. accommodation. In Model 1, both E-site and P-site tRNAs slip into the +1 frame and follow the +1 frame aa-tRNA selection to produce frameshifted proteins (P1A1). In Model 1A, both E-site and P-site tRNAs are destabilized by stimulatory signals and follow the +1 frame aa-tRNA selection to produce frameshifted proteins (P1A1). Models 2 and 3 differ in the timing of the E-site tRNA dissociation step (in Model 2, E-site tRNA dissociation occurs during the codon recognition step while in Model 3, E-site tRNA dissociates after codon recognition). Both Models 2 and 3 result in the formation of ribosomes with only P-site tRNA (P0), which can slip to the +1 frame to form P1 and result in the formation of frameshifted proteins (P1A1).
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Figure 1: The three kinetic models for +1 PRF in E. coli. Steps I–VI illustrate the non-frameshifting translation elongation process: I. initial binding; II codon recognition; III. GTPase activation; IV. GTP hydrolysis; V. EF-Tu dissociation; VI. accommodation. In Model 1, both E-site and P-site tRNAs slip into the +1 frame and follow the +1 frame aa-tRNA selection to produce frameshifted proteins (P1A1). In Model 1A, both E-site and P-site tRNAs are destabilized by stimulatory signals and follow the +1 frame aa-tRNA selection to produce frameshifted proteins (P1A1). Models 2 and 3 differ in the timing of the E-site tRNA dissociation step (in Model 2, E-site tRNA dissociation occurs during the codon recognition step while in Model 3, E-site tRNA dissociates after codon recognition). Both Models 2 and 3 result in the formation of ribosomes with only P-site tRNA (P0), which can slip to the +1 frame to form P1 and result in the formation of frameshifted proteins (P1A1).

Mentions: An elegant series of biochemical studies have contributed to a very detailed kinetic model of A-site tRNA selection (19). In this model, fast initial binding of the ternary complex EF-Tu:aa-tRNA:GTP is followed by codon recognition. Codon recognition triggers EF-Tu GTPase activation, which leads to the GTP hydrolysis and dissociation of EF-Tu from the ribosome. Factor dissociation is followed by the spontaneous accommodation of the acceptor end of the aa-tRNA into the A-site or the rejection of the aa-tRNA by proofreading. This concept is illustrated along the top of Figure 1.Figure 1.


A new kinetic model reveals the synergistic effect of E-, P- and A-sites on +1 ribosomal frameshifting.

Liao PY, Gupta P, Petrov AN, Dinman JD, Lee KH - Nucleic Acids Res. (2008)

The three kinetic models for +1 PRF in E. coli. Steps I–VI illustrate the non-frameshifting translation elongation process: I. initial binding; II codon recognition; III. GTPase activation; IV. GTP hydrolysis; V. EF-Tu dissociation; VI. accommodation. In Model 1, both E-site and P-site tRNAs slip into the +1 frame and follow the +1 frame aa-tRNA selection to produce frameshifted proteins (P1A1). In Model 1A, both E-site and P-site tRNAs are destabilized by stimulatory signals and follow the +1 frame aa-tRNA selection to produce frameshifted proteins (P1A1). Models 2 and 3 differ in the timing of the E-site tRNA dissociation step (in Model 2, E-site tRNA dissociation occurs during the codon recognition step while in Model 3, E-site tRNA dissociates after codon recognition). Both Models 2 and 3 result in the formation of ribosomes with only P-site tRNA (P0), which can slip to the +1 frame to form P1 and result in the formation of frameshifted proteins (P1A1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2377451&req=5

Figure 1: The three kinetic models for +1 PRF in E. coli. Steps I–VI illustrate the non-frameshifting translation elongation process: I. initial binding; II codon recognition; III. GTPase activation; IV. GTP hydrolysis; V. EF-Tu dissociation; VI. accommodation. In Model 1, both E-site and P-site tRNAs slip into the +1 frame and follow the +1 frame aa-tRNA selection to produce frameshifted proteins (P1A1). In Model 1A, both E-site and P-site tRNAs are destabilized by stimulatory signals and follow the +1 frame aa-tRNA selection to produce frameshifted proteins (P1A1). Models 2 and 3 differ in the timing of the E-site tRNA dissociation step (in Model 2, E-site tRNA dissociation occurs during the codon recognition step while in Model 3, E-site tRNA dissociates after codon recognition). Both Models 2 and 3 result in the formation of ribosomes with only P-site tRNA (P0), which can slip to the +1 frame to form P1 and result in the formation of frameshifted proteins (P1A1).
Mentions: An elegant series of biochemical studies have contributed to a very detailed kinetic model of A-site tRNA selection (19). In this model, fast initial binding of the ternary complex EF-Tu:aa-tRNA:GTP is followed by codon recognition. Codon recognition triggers EF-Tu GTPase activation, which leads to the GTP hydrolysis and dissociation of EF-Tu from the ribosome. Factor dissociation is followed by the spontaneous accommodation of the acceptor end of the aa-tRNA into the A-site or the rejection of the aa-tRNA by proofreading. This concept is illustrated along the top of Figure 1.Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.

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.

Show MeSH
Related in: MedlinePlus