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Transit of tRNA through the Escherichia coli ribosome: cross‐linking of the 3′ end of tRNA to ribosomal proteins at the P and E sites
Author(s) -
Kirillov Stanislav V,
Wower Jacek,
Hixson Stephen S,
Zimmermann Robert A
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)02302-5
Subject(s) - transfer rna , ribosome , t arm , p site , 50s , a site , 23s ribosomal rna , ribosomal protein , ribosomal rna , eukaryotic ribosome , biochemistry , 30s , escherichia coli , biology , rna , binding site , chemistry , gene
Photoreactive derivatives of yeast tRNA Phe containing 2‐azidoadenosine at their 3′ termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)‐programmed ribosomes, deacylated tRNA Phe , Phe‐tRNA Phe and N ‐acetyl‐Phe‐tRNA Phe probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA Phe bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA Phe probe also labeled protein L1. Cross‐linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.