A paradigm for local conformational control of fucntion in the ribosome: binding of ribosomal protein S19 to Escherichia coli 16S rRNA in the presence of S7 is required for methylation of m 2 G966 and blocks methylation of m 5 C967 by their respective methyltransferases (original) (raw)

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Roche Institute of MOlecular Biology, Roche Research Center

Nutley, NJ 07110, USA

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Roche Institute of MOlecular Biology, Roche Research Center

Nutley, NJ 07110, USA

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Roche Institute of MOlecular Biology, Roche Research Center

Nutley, NJ 07110, USA

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Roche Institute of MOlecular Biology, Roche Research Center

Nutley, NJ 07110, USA

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Published:

25 December 1991

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Carl Weitzmann, Santa J. Tumminia, Miloslav Boublik, James Ofengand, A paradigm for local conformational control of fucntion in the ribosome: binding of ribosomal protein S19 to Escherichia coli 16S rRNA in the presence of S7 is required for methylation of m 2 G966 and blocks methylation of m 5 C967 by their respective methyltransferases , Nucleic Acids Research, Volume 19, Issue 25, 25 December 1991, Pages 7089–7095, https://doi.org/10.1093/nar/19.25.7089
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Abstract

We have partially purified two 16S rRNA-specific methyltransferases, one of which forms m 2 gse ( m 2 GMT), while the other one makes m 5 C967 (m 5 C RAT). The m 2 G RAT uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA, while the m 5 C RflT functions reciprocally, using free rRNA but not 30S subunits (Negre, D., Weitzmann, C. and Ofengand, J. (1990) UCLA Symposium: Nucleic Acid itfethylation (Alan Liss, New York), pp. 1–17). We have now determined the basis for this unusual inverse specificity at adjacent nucleotides. Binding of ribosomal proteins S7, S9, and S19 to unmodified 16S rRNA individually and in all possible combinations showed that S7 plus S19 were sufficient to block methylation by the m 5 C RAT, while simultaneously inducing methylation by the m 2 G RAT. A purified complex containing stoichiometric amounts of proteins S7, S9, and S19 bound to 16S rRNA was isolated and shown to possess the same methylation properties as 30S subunits, that is, the ability to be methylated by the m 2 G RAT but not by the m 5 C RAT. Since binding of S19 requires prior binding of S7, which had no effect on methylation when bound alone, we attribute the switch in methylase specificity solely to the presence of RNA-bound S19. Single-omission reconstitute of 30S subunits deficient in S19 resulted in particles that could not be efficiently methylated by either enzyme. Thus while binding of S19 is both necessary and sufficient to convert 16S rRNA into a substrate of the m 2 G RAT, binding of either S19 alone or some other protein or combination of proteins to the 16S rRNA can abolish activity of the m 5 C MT. Binding of S19 to 16S rRNA is known to cause local conformational changes in the 960–975 stem-loop structure surrounding the two methylated nucleotides (Powers, T., Changchien, L.-RA., Craven, G. and Noller, H.F. (1988) J. Mol. Biol . 200, 309–319). Our results show that the two ribosomal RNA RflTs studied In this work are exquisitely sensitive to this small but nevertheless functionally Important structural change.

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