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Molecular and Cellular Biology, January 1999, p. 826-834, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Ribosomal Protein S14 of Saccharomyces
cerevisiae Regulates Its Expression by Binding to
RPS14B Pre-mRNA and to 18S rRNA
Sheara W.
Fewell and
John L.
Woolford Jr.*
Department of Biological Sciences, Carnegie
Mellon University, Pittsburgh, Pennsylvania 15213
Received 29 July 1998/Returned for modification 26 August
1998/Accepted 24 September 1998
Production of ribosomal protein S14 in Saccharomyces
cerevisiae is coordinated with the rate of ribosome assembly by a
feedback mechanism that represses expression of RPS14B.
Three-hybrid assays in vivo and filter binding assays in vitro
demonstrate that rpS14 directly binds to an RNA stem-loop structure in
RPS14B pre-mRNA that is necessary for RPS14B
regulation. Moreover, rpS14 binds to a conserved helix in 18S rRNA with
approximately five- to sixfold-greater affinity. These results support
the model that RPS14B regulation is mediated by direct
binding of rpS14 either to its pre-mRNA or to rRNA. Investigation of
these interactions with the three-hybrid system reveals two regions of
rpS14 that are involved in RNA recognition. D52G and E55G mutations in
rpS14 alter the specificity of rpS14 for RNA, as indicated by increased
affinity for RPS14B RNA but reduced affinity for the rRNA
target. Deletion of the C terminus of rpS14, where multiple antibiotic
resistance mutations map, prevents binding of rpS14 to RNA and
production of functional 40S subunits. The emetine-resistant protein,
rpS14-EmRR, which contains two mutations near the C
terminus of rpS14, does not bind either RNA target in the three-hybrid
or in vitro assays. This is the first direct demonstration that an
antibiotic resistance mutation alters binding of an r protein to rRNA
and is consistent with the hypothesis that antibiotic resistance
mutations can result from local alterations in rRNA structure.
*
Corresponding author. Mailing address: Department of
Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213. Phone: (412) 268-3193. Fax: (412) 268-7129. E-mail:
JW17{at}andrew.cmu.edu.
Molecular and Cellular Biology, January 1999, p. 826-834, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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