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Abstract: Ribosomal protein S20 is a primary binding protein that bridges the 5′ domain and the 3′ minor domain of the 16S ribosomal RNA (rRNA) in the 30S ribosomal subunit. Using time-dependent dimethyl sulfate modification, we have determined that as it is bound to 16S rRNA, protein S20 causes rapid protection of bases A246, A274, A279, and A282 in the stem region of helix 11 in the 5′ domain and moderately fast modifications of helix 44 bases A1433 and A1434 in the 3′ minor domain. At a later time, enhancements occur with bases A181and A190 in helix 9, bases A325 and A327 in helix 13, and base C264 at the distal end of helix 11 in the 5′ domain of 16S rRNA. The modifications that occur in the stem region of helix 11 are distant from the binding site of protein S20, as determined from the crystal structure. Simultaneous addition of protein S17 with S20 to the complex significantly alters the modifications caused by protein S20 in the stem region of helix 11 but does not alter the remaining modifications. Our results indicate that protein S20 is binding to at least two alternate 16S rRNA sites during the early assembly process. [ABSTRACT FROM AUTHOR]

Abstract: Ribosomal subunit assembly is initiated by the binding of several primary binding proteins. Results from chemical modification studies show that 16S ribosomal RNA undergoes striking structural rearrangements when protein S17 is bound. For the first time, we are able to distinguish and order these structural rearrangements by using time-dependent chemical probing. Initially, protein S17 binds to a portion of helix 11, inducing a kink-turn in that helix that bends helix 7 toward the S17–helix 11 complex in a hairpin-like manner, allowing helix 7 to bind to protein S17. This structural change is rapidly stabilized by interactions at the distal and proximal ends of both RNA helices. Identifying the dynamic nature of interactions between RNA and proteins is not only essential in unraveling ribosome assembly, but also has more general application to all protein–RNA interactions. [Copyright &y& Elsevier]

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]

The notion that the ribosome is dynamic has been supported by various biochemical techniques, as well as by differences observed in high-resolution structures of ribosomal complexes frozen in various functional states. Yet, the mechanisms and extent of rRNA dynamics are still largely unknown. We have used a novel, fast chemical-modification technique to provide time-resolved details of 16S rRNA structural changes that occur as bridges are formed between the ribosomal subunits as they associate. Association of different 16S rRNA regions was found to be a sequential, multi-step process involving conformational rearrangements within the 30S subunit. Our results suggest that key regions of 16S rRNA, necessary for decoding and tRNA A-site binding, are structurally altered in a time-dependent manner by association with the 50S ribosomal subunits. [Copyright &y& Elsevier]