Crosslinking of N‐Acetyl‐phenylalanyl [s⁴U]tRNA^Phe to Protein S10 in the Ribosomal P Site

Abstract

In order to identify ribosomal components involved in the peptidyl-tRNA binding site on the ribosome, tRNAPh' molecules were prepared in which cytidine residues had been chemically converted into 4-thiouridine (s"U). This nucleoside is photoactive at 335 nm and able to form covalent bonds with nearby nucleophilic groups. The thiolated AcPhe-tRNAPhe was bound to the ribosomal P site in the presence of poly(U) as verified by puromycin reactivity. Direct irradiation of the AcPhe-[s4U]tRNAPhe/po1y(U)/70-S ribosome complex induced crosslinking of the tRNA molecule exclusively to 30-S subunits. Analysis of the covalent complex revealed that AcPhe-[s4U]tRNAPh' was specifically crosslinked to protein SIO.Protein biosynthesis is based on a coordinated interaction among several macromolecules including mRNA, tRNAs and translational factors on the ribosome. To achieve this complex function, the ribosomal particle possesses specific binding sites for the various ligands. Understanding the functional role of the ribosome requires the identification and localization of components involved in each step of protein synthesis. Although it is widely accepted that tRNA has at least two binding sites on the ribosome, the Asite (for aminoacyl-tRNA) and the P site (for peptidyl-tRNA), little is known about the molecular mechanisms of tRNA attachment. In spite of numerous data accumulated over the past 15 years, unambiguous localization of both sites on the ribosomal particle has yet to be achieved.Several approaches have been developed to study the topography of tRNA-ribosome complexes such as immunoelectron microscopy [I, 21, affinity and photoaffinity labelling [3,4], and a combination of affinity labelling and immunoelectron microscopy [5].In order to characterize ribosomal tRNA binding sites more fully, we prepared a photoactive derivative of tRNAPhe. The methodology involves a chemical conversion of cytidine residues within tRNAPhe into 4-thiouridine (s4U) by treatment with HZS. The s4U residue can form covalent bonds with either nucleotides [6] or proteins [7] located in its close vicinity upon irradiation at 335 nm.In this paper we focus on the ribosomal P site binding region. We report that thiolated AcPhe-tRNAPhe is crosslinked to the 30-S subunit within the 70-S ribosome. All the crosslinked tRNA molecules are associated with protein SIO. MATERIALS AND METHODS MaterialsPure tRNAPh' was prepared from brewer's yeast tRNA (Boehringer, Mannheim) by counter-current distribution according to Dirheimer and Ebel [8] followed by chromatography on benzoylated DEAE-cellulose. The material was eluted with a combined gradient of NaCl (0.85 -1.2 M) and dimethylformamide (0-4%) in 5 mM sodium formate pH 4.0,20 mM MgCIz.Purified yeast phenylalanyl-tRNA synthetase and yeast tRNA nucleotidyltransferase were prepared in our laboratory as described by Fasiolo et al.

Suppressor mutations in ribosomal proteins and FliY restore Bacillus subtilis swarming motility in the absence of EF-P

“…Rae1 has been recently shown to act as a ribosomal A-site endoribonuclease, and it was hypothesized that ribosome stalling may increase its access to its mRNA substrate and thereby increase its activity [42]. Finally, S3 and S10 are components of the small subunit of the ribosome itself: S3 is involved in mRNA processivity and S10 is involved in binding to the P-site tRNA [48,50]. Further, the residue altered by soe24 (S10 M88R ) has been implicated in the direct interaction with the last protein identified by efp suppressor analysis, NusG [49].…”