To gain a better understanding of the architecture of the ribosome in general, and the structure of the ribosome within the tRNA binding sites specifically, photoreactive tRNA derivatives were cross-linked to the Escherichia coli ribosome. Protein and RNA components in the immediate vicinity of the tRNA were identified by cross-linking yeast tRNA$\sp{\rm Phe}$ molecules, substituted with the photoreactive nucleoside 2-azidoadenosine at positions 37, 73 or 76, to the ribosomal tRNA binding sites. These tRNA derivatives were good substrates for aminoacyl-tRNA synthetase and were specifically bound and cross-linked to the A (aminoacyl), P (peptidyl) and E (exit) sites on the ribosome. Yeast tRNA$\sp{\rm Phe}$, containing 2-azidoadenosine at position 37, was used to probe the decoding domain on the 30S subunit since the photoreactive base was immediately adjacent to the 3$\sp\prime$ end of the anticodon. From the A, P and E sites, cross-linking was exclusively to the 30S subunit and both proteins and 16S rRNA were labeled. While protein S7 was cross-linked in the A and P sites, protein S11 was labeled in the E site. The 16S rRNA nucleotides C1317 and C1359 were labeled in the P site, and U1135, C1226, C1228, C1237, C1249 and C1284 in the A site. In addition, the E site-bound tRNA cross-linked nucleotide(s) within the 3$\sp\prime$ terminal 29 bases of the 16S rRNA. While the tRNA-16S rRNA cross-links in the A and P sites are generally consistent with the current three-dimensional models of the 3$\sp\prime$ major domain, they suggest that this region of the 16S rRNA should be positioned closer to nucleotide C1400, which was previously cross-linked to the 5$\sp\prime$ anticodon base of P site-bound tRNA$\sb1\sp{\rm Val}$. Since several immune electron microscopy studies place C1400 deep in the cleft of the 30S subunit, both the 16S rRNA nucleotides and protein S7, cross-linked in this study, must also be associated with this unique topographical feature. In the E site, the anticodon loop is proposed to reside on the platform of the 30S subunit, since both the 3$\sp\prime$ end of 16S rRNA and protein S11 have been located in this region by immune electron microscopy. When tRNA$\sp{\rm Phe}$ derivatives containing 2-azidoadenosine at either position 73 or 76 were bound and cross-linked to the A site, protein L27 was the main target of labeling. The cross-linking of protein L27 by azidoadenosines at or near the aminoacyl-end of tRNA in the A site adds to a mounting body of evidence suggesting that this protein is a central component of the peptidyl transferase center. While no ribosomal RNA was labeled by the tRNA probe substituted at position 73, significant labeling of 23S rRNA occurred when the derivative substituted at position 76 was cross-linked to the A site.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8520 |
| Date | 01 January 1992 |
| Creators | Sylvers, Lee Alan |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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