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Abstract
Ribosomal protein S4 protects a characteristic set of bases in 16 S rRNA from attack by chemical probes. Use of hydroxyl radical as a probe of the RNA backbone shows that ribose residues in these same regions are also protected by S4, confirming the localization of its interactions with 16 S rRNA to the junction of five helical elements in the proximal region of the 5' domain. At 0 degrees C, the nucleotides protected by S4 from base-specific probes are confined almost exclusively to the two compound helices formed by residues 404-499. After subsequent heating of the complex briefly at 30 or 42 degrees C, nucleotides in the three adjacent helices are additionally protected, resulting in a pattern of protection that is identical to that which is observed when S4 is incubated with 16 S rRNA under in vitro reconstitution conditions. Preincubation of the protein or the RNA (or both) separately at elevated temperature does not substitute for heating the S4.RNA complex. The regions in the RNA affected by the heat step are known to interact with proteins S12 and S16, both of which depend upon S4 for their binding to the RNA. Thus, the finding that S4 recruits additional sites of interaction in the RNA following its initial binding suggests a possible mechanism to insure the sequential addition of proteins during ribosomal assembly.
Highlights
Institutes of Health and by a grant to the Center for MolecularBiology of RNA from the Lucille P
The protected bases were confined to a limited region of the domain comprising the junction of five helical elements, which was termed the "84 junction." This region of the RNA lies within the RNase-resistant fragments characterized in the earlier nuclease protection studies, but it defines a compact RNA substructure that could more plausibly constitute the actual binding site for a protein of the size of 84
The results of hydroxyl radical protection, which probes interactions involving the RNA backbone, strongly support the conclusions from our earlier studies; all of the 84dependent protections ofthe RNA backbone are localized to the 84 junction region
Summary
Vol 270, No., Issue of January 20, pp. 1238-1242, 1995 Printed in U.S.A. A Temperature-dependent Conformational Rearrangement in the Ribosomal Protein 84·16 8 rRNA Complex*. Direct cross-linking of 84 to one of the helices that forms the 84 junction supports this assignment (Greuer et al, 1987) Using another approach, Vartikar and Draper (1988) studied the RNA binding site requirements for protein 84 by constructing synthetic subfragments of 16 8 rRNA using in vitro transcription and testing the ability of the synthetic rRNA analogues to bind 84. Vartikar and Draper (1988) studied the RNA binding site requirements for protein 84 by constructing synthetic subfragments of 16 8 rRNA using in vitro transcription and testing the ability of the synthetic rRNA analogues to bind 84 They found that deletion of regions of the RNA containing two of the five implicated helical elements had little or no effect on the specific binding of 84 to the RNA, in apparent contradiction to our findings. The ability of protein 84 to participate in two distinct and separable interactions with different structural features of 168 rRNA raises new questions concerning mechanisms of RNA-protein interactions in the ribosome
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