Structure and mechanism of 16SrRNA in the ribosome studied by photocrosslinking.

摘要

The ribosome is a complex RNA-protein machine that translates the genetic information contained in the mRNA molecule to a sequence of amino acids. This involves mediating the decoding of the mRNA sequence, peptide bond formation and translocation. Throughout the process of translation, the ribosome undergoes significant conformational changes. The crystal structures of ribosome and the ribosomal subunits have been resolved at moderate resolution, but the details of conformational changes at various steps of translation have just started to be understood. Both structural and biochemical techniques have been applied towards the understanding of the ribosome dynamics. UVB (far UV, 210--300 nm) radiation has been used to study the structural changes in 16S rRNA within the 30S ribosomal subunit by monitoring the changes in the crosslinking frequencies of RNA-RNA crosslinks. In the first part of this study additional RNA crosslinks in 16S rRNA are determined after in vivo incorporation of 4-thiouridine (s4U) into RNA in a strain of Escherichia coli deficient in pyrimidine synthesis followed by irradiation with UVA (near UV, 300--380 nm) light. Similar crosslinks are observed when synthetic 16S rRNA transcribed with s 4U is reconstituted into 30S subunits except that there are a few conformational differences between in vivo and in vitro synthesized subunits. Crosslinks obtained from UVB and s 4U-UVA irradiation show a striking similarity between their identity and location. All the crosslinks are found to be restricted in the 30S subunit making a reversed "C" shape through the center of the subunit. These results predict local RNA conformational flexibility that is most likely the factor in determining the formation of the crosslinks. In the second part, the effects of initiation factors on the structural status of the 30S initiation complex are studied. The results indicate IF3 is the main player in determining the 30S structure during initiation and that its C-domain (IF3C) causes similar structural changes in the 16S rRNA.