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Chapter 20 : Reconstitution of the 50S Subunit with In Vitro-Transcribed 23S rRNA: a New Tool for Studying Peptidyltransferase

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Reconstitution of the 50S Subunit with In Vitro-Transcribed 23S rRNA: a New Tool for Studying Peptidyltransferase, Page 1 of 2

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Abstract:

RNA plays a critical role in all the steps of protein synthesis. The idea that the modern ribosome evolved from the original RNA-only protein-synthesizing machinery was so appealing, and made so much sense from the evolutionary standpoint, that it immediately found many proponents. The high degree of rRNA sequence conservation is usually perceived as an indication of the functional importance of the corresponding rRNA segment. Several nucleotide sequence segments of 23S rRNA, as well as a number of individual nucleotides, are invariant among all the studied organisms. Peptidyltransferase substrates, aminoacyl- and peptidyl-tRNAs, form tight contacts with rRNA, which has been demonstrated most clearly by crosslinking and footprinting experiments. By contrast, the omission of 5S rRNA during reconstitution of 50S subunits with either natural or in vitro-transcribed 23S rRNA had a significantly more severe effect on the peptidyltransferase activity, which was reduced to the level of no more than 0.03% of that of native 50S subunits. The RNA component of the peptidyltransferase center is composed of RNA segments scattered in the 23S rRNA primary structure. While some rRNA sequences are brought together in the rRNA secondary structure, other elements are brought into proximity with the catalytic center in the tertiary structure of the 50S subunit.

Citation: Khaitovich P, Mankin A. 2000. Reconstitution of the 50S Subunit with In Vitro-Transcribed 23S rRNA: a New Tool for Studying Peptidyltransferase, p 229-243. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch20

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Figures

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Figure 1

Two-dimensional gel electrophoresis of proteins from 50S subunits and KSP particles. The identified proteins of KSP particles are labeled. Spot X in protein preparations from KSP particles was not sequenced; the corresponding spot in total protein of the 50S subunit contains unresolved proteins L1 and L4.

Citation: Khaitovich P, Mankin A. 2000. Reconstitution of the 50S Subunit with In Vitro-Transcribed 23S rRNA: a New Tool for Studying Peptidyltransferase, p 229-243. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch20
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Image of Figure 2
Figure 2

Reconstitution of 50S subunits with natural and in vitro-transcribed 23S rRNA. (A) Temperature profiles for the two-step and three-step reconstitutions. (B) Sucrose gradient analysis of native 50S subunits or subunits reconstituted with natural or in vitro-transcribed 23S rRNA. The arrows indicate the positions of the peaks of native 50S subunits. (C) Peptidyltransferase activity of subunits reconstituted with in vitro-transcribed 23S rRNA in a two- or threestep reconstitution procedure. The spots shown correspond to the product of the peptidyltransferase reaction, [S]fMetpuromycin, resolved by paper electrophoresis (for details, see ). The histogram at the bottom of panel C shows the results of quantification of the [S]fMet-puromycin spot (total counts over a 10-h exposure).

Citation: Khaitovich P, Mankin A. 2000. Reconstitution of the 50S Subunit with In Vitro-Transcribed 23S rRNA: a New Tool for Studying Peptidyltransferase, p 229-243. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch20
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Figure 3

Compensatory effect of G2553C mutation in 23S rRNA and C75G mutation in aminoacyl-tRNA or its analogues. The schematic secondary structure of domain V of 23S rRNA and the position of G2553 are shown on the left. (A) Peptidyltransferase reaction between [S]fMet-tRNA and puromycin-containing oligonucleotides, CC-Pm or CGPm, catalyzed by 50S subunits reconstituted with the wild-type (G2553) or mutant (C2553) 23S rRNA. The reaction product, [S]fMet-puromycin, was resolved by paper electrophoresis and quantified. (B) Effect of G2553C mutation in 23S rRNA on peptidyltransferase reaction between [S]fMet-tRNA and either wild-type (C75) or mutant (G75) Val-tRNA ( ). The reaction product, [S]fMet-Val dipeptide, was resolved by thin-layer chromatography. In both experiments, the amount of radioactivity in the reaction product, [S]fMet-puromycin or [S]fMet-Val, was quantified after a 10-h exposure on a beta-scanner.

Citation: Khaitovich P, Mankin A. 2000. Reconstitution of the 50S Subunit with In Vitro-Transcribed 23S rRNA: a New Tool for Studying Peptidyltransferase, p 229-243. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch20
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Figure 4

Effect of 5S rRNA and the ketolide antibiotic HMR3647 on the reconstitution of catalytically active large ribosomal subunits. (A) Sites of interactions of 5S rRNA and some macrolide, ketolide, and streptogramin B-type antibiotics with 23S rRNA. The regions boxed by solid lines in domains II and V show the sites of occurrence of antibiotic resistance mutations and/or rRNA positions whose accessibilities to chemical modifications are affected by the antibiotics. The region in domain V boxed by a dashed line shows the locations of 23S rRNA positions protected from chemical modification upon binding of the 5S rRNA-protein complex. 5S rRNA-23S rRNA cross-links are shown by dashed lines (see the text for references). (B and C) Effect of the presence of a ketolide, HMR3647, during the reconstitution procedure on the peptidyltransferase activity of 50S subunits assembled without 5S rRNA. The spots in panel C represent the product of the peptidyltransferase reaction, [S]fMet-puromycin. The drug was added before the first incubation step (lane 1), before the third incubation step (lane 2), or after completion of reconstitution (lane 3), as also shown in panel B. +, present; -, absent.

Citation: Khaitovich P, Mankin A. 2000. Reconstitution of the 50S Subunit with In Vitro-Transcribed 23S rRNA: a New Tool for Studying Peptidyltransferase, p 229-243. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch20
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