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Chapter 31 : The Unbearable Lightness of Peptidyl-tRNA

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The Unbearable Lightness of Peptidyl-tRNA, Page 1 of 2

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Abstract:

In the conventional picture of protein synthesis, the ribosome advances by 3 nucleotides along the mRNA in each translocation step—or, looked at differently, the mRNA with peptidyl-tRNA associated threads back through the ribosome by 3 nucleotides. In either case, triplet movement is assumed to be the natural state of affairs. It has been clear for nearly 2 decades that the interaction of mRNA with ribosomes and with tRNAs was compatible with nontriplet movement. This chapter concentrates on the “shifty” sequence in the immediate region of the frameshift event in relationship to certain limited aspects of ribosome behavior. Starvation-induced frameshifting falls into the category of peptidyl-tRNA slippage. The authors are left with the implication that noncognate interactions in the A site have little to do with frame shifting, while slippage of peptidyl-tRNA in the P site has everything to do with it. In fact, the mobility of peptidyl-tRNA is illustrated in much more dramatic fashion by two other related phenomena. If cognate peptidyl-tRNA exhibits some nonzero probability per unit time of slipping, even if this probability is very small, aminoacyl-tRNA limitation will necessarily increase the time during which such a slip may occur and thereby increase its overall probability of occurring. A variety of data demonstrate that certain tRNA modifications in the anticodon loop and stem affect the tendency of certain tRNA species to frameshift.

Citation: Gallant J, Lindsley D, Masucci J. 2000. The Unbearable Lightness of Peptidyl-tRNA, p 385-396. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch31

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Figures

Image of Figure 1
Figure 1

Amino acid sequence in the region of a leftward (-1) ribosome frameshift at a hungry codon. The protein was encoded by a left-frameshift reporter; the promoter was induced, and cells were subjected to partial inhibition by isoleucine-hydroxamate, which stimulated synthesis of frameshifted, active enzyme to about 10% that of a zero-frame control; the resulting -galactosidase was purified and cleaved specifically, and the fragment with the indicated valine residue at the N terminus was submitted to Edman N-terminal sequencing. The PTH-amino acids released in successive cycles are shown in conventional, cycle-bar plot form. The messenger nucleotide sequence is shown below the cycle-bar plots. The amino acids encoded in the zero-frame are shown above the nucleotide sequence; those corresponding to the leftward (-1) frame after the shift (arrow) are shown below the nucleotide sequence. (From )

Citation: Gallant J, Lindsley D, Masucci J. 2000. The Unbearable Lightness of Peptidyl-tRNA, p 385-396. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch31
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Image of Figure 2
Figure 2

Amino acid sequence in the region of a rightward (+1) ribosome frameshift at a hungry codon. The protein was encoded by a right-frameshift reporter; cells were induced and inhibited by isoleucine-hydroxamate as for Fig. 1 , stimulating enzyme synthesis 40-fold to 27% that of the parallel, zero-frame control; purification, cleavage, and sequencing were done as for Fig. 1 . The nucleotide sequence is shown as the coding strand of the DNA; the predicted amino acid sequences in all three reading frames are shown below the nucleotide sequence, with the limiting isoleucine in the zero-frame position 7 in italics; the observed amino acid sequence, which shifts rightward at this position, is shown in boldface letters.

Citation: Gallant J, Lindsley D, Masucci J. 2000. The Unbearable Lightness of Peptidyl-tRNA, p 385-396. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch31
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Image of Figure 3
Figure 3

Efficiency of slides of 7 or 40 nucleotides induced at a hungry AUA codon. Constructs L-7 and L-40 (see ) demand that the ribosome•peptidyl-tRNA complex slide downstream 7 or 40 nucleotides, respectively, from a hungry AUA codon to find a landing site beyond blocking terminators and thus produce -galactosidase. The relevant region of the messenger sequence is, in schematic form, UUC AUA (7 or 40 nucleotides ending in UAA) UUU →[ reading frame]. The differential rate of enzyme synthesis during one doubling of partial isoleucine-tRNA limitation is plotted on the axis versus the concentration of isoleucine-hydroxamate (ILHX). Note the two different scales on the axis for the slide of 7 (at left) and the slide of 40 (at right) nucleotides.

Citation: Gallant J, Lindsley D, Masucci J. 2000. The Unbearable Lightness of Peptidyl-tRNA, p 385-396. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch31
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Image of Figure 4
Figure 4

Kinetics of frameshift induction during isoleucine-tRNA limitation in a - mutant isogenic pair. The left-frameshift reporter is similar to those described by , with the following shifty sequence in the message: U UUU AUA. Log-phase cells were induced, and a subculture was subjected to isoleucine-hydroxamate (ILHX) inhibition (72 μg/ml) and sampled over a period of about two doublings. The data are shown in the form of a differential plot of enzyme synthesis versus increase in protein. Open circles, control, uninhibited subcultures; solid circles, ILHX-inhibited subcultures. (A) CP78 ( ); (B) CP79 ( mutant). The inset shows the first four points of the ILHX-inhibited culture on an expanded scale. In the data for CP79 in ILHX, note the lag period and the subsequent enormous acceleration of the differential rate.

Citation: Gallant J, Lindsley D, Masucci J. 2000. The Unbearable Lightness of Peptidyl-tRNA, p 385-396. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch31
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