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Abstract: The penultimate stem–loop of 16S ribosomal RNA (rRNA), helix 44, plays a central role in ribosome function. Using time-resolved dimethyl sulfate (DMS) probing, we have analyzed time-dependent modifications that occur at specific bases in this helix near the decoding region, resulting from the binding of elongation factor G (EF-G) in various forms. When EF-G-GTP is bound to 70S ribosomes, bases A1492 and A1493 are immediately protected, while other bases in the region show either no change or enhanced modification. When apo-EF-G is bound to 70S ribosomes and GTP is added, substantial transient time-dependent enhancement occurs at bases A1492 and A1493, with somewhat less enhancement occurring at base A1483, all in the first 45 ms. When mRNA and deacylated tRNAs are bound to the 70S ribosome and EF-G-GTP is added, bases A1492 and A1493 again show substantial and continued enhancement, while bases A1408, A1413, and A1418 all show time-dependent protection. These results provide primary, real-time evidence that EF-G induces direct or indirect structural changes in this region as EF-G is bound and as GTP is hydrolyzed. [Copyright &y& Elsevier]

Abstract: Time-dependent chemical modification and cleavage results have provided intriguing insights into structural changes that occur in the distal loop of helix 11 in 16S ribosomal RNA (rRNA). Located distant from the decoding region, between proteins S17 and S20, the results of this study suggest that this region of rRNA may act as a buffer or a spring between these two proteins during protein biosynthesis. During the assembly process, protein S17 apparently produces the major structural deformations in this region, causing it to be folded in a spring-like structure. Base C264 in this region shows erratic behavior during assembly and also shows time-dependent enhancement when elongation factor G with GTP is added to 70S ribosomes. Evidence is presented to suggest that this region of rRNA may be used to allow relative motion to occur between proteins S17 and S20 during translocation. [Copyright &y& Elsevier]