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

Abstract 

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.

Keywords:  Photoaffinity labeling, 2-Azidoadenosine, Proteins L1, L27, L33, Protein synthesis

• 1 Permanent address: Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina 188350, Leningrad Region, Russia.
• 2 Present address: Department of Animal Sciences, Auburn University, Auburn, AL 36849, USA.