Alteration in Location of a Conserved GTPase-associated Center of the Ribosome Induced by Mutagenesis Influences the Structure of Peptidyltransferase Center and Activity of Elongation Factor G

Abstract

Translocation catalyzed by elongation factor G occurs after the peptidyltransferase reaction on the large ribosomal subunit. Deacylated tRNA in the P-site stimulates multiple turnover GTPase activity of EF-G. We suggest that the allosteric signal from the peptidyltransferase center that activates EF-G may involve the alteration in the conformation of elongation factor binding center of the ribosome. The latter consists of the moveable GTPase-associated center and the sarcin-ricin loop that keeps its position on the ribosome during translation elongation. The position of the GTPase-associated center was altered by mutagenesis. An insertion of additional base pair at positions C1030/G1124 was lethal and affected function of EF-G, but not that of EF-Tu. Structure probing revealed a putative allosteric signal pathway connecting the P-site with the binding site of the elongation factors. The results are consistent with the different structural requirements for EF-G and EF-Tu function, where the integrity of the path between the peptidyltransferase center and both GTPase-associated center and sarcin-ricin loop is important for EF-G binding.The ribosome is a large molecular machine for protein synthesis. During the elongation EF 1 -Tu brings aminoacyl-tRNA to the ribosomal A-site, and its GTPase activity is stimulated by recognition of the mRNA codon by the aminoacyl-tRNA anticodon at the decoding center of the small subunit. EF-G acts after completion of peptide transfer at the peptidyltransferase center of the large subunit.Before the peptidyltransferase reaction, the growing peptide is attached to the 3Ј-end of the tRNA bound to the P-site, whereas the aminoacyl-tRNA is located in the A-site. After peptide transfer, the A-site carries the peptidyl-tRNA, whereas the P-site holds the deacylated tRNA. This is the state that should be recognized by EF-G. It has been reported that deacylated tRNA in the P-site stimulates multiple turnover GTPase activity of EF-G, but that peptidyl-tRNA does not (1, 2). This stimulation is attributed to the enhanced binding of EF-G to the ribosome (2).The P-site is located more than 70 Å away from the binding site of the elongation factors. Thus, an intriguing question is how can information from the P-site be transmitted across half the width of the ribosome? We believe that to answer this question one must seek for elements that are located between the peptidyltransferase center and the elongation factor binding site, the latter consisting of the GTPase-associated center (GAC) and the sarcin-ricin loop (SRL) (3). One can expect that some of the relevant elements should be mobile, as they have to transfer an allosteric signal by its movement relative to the rest of the large subunit. The SRL interacts with the switch regions of the elongation factors close to the GTP binding site (4), and it is likely that it is involved in the activation of GTP hydrolysis. However, in all available atomic structures of large ribosomal subunits from various organisms, as well as in cryoelectron microscop...

The sarcin–ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit

“…The base reactivity changes we observe in the DSRL 50S subunits show extensive overlap with those in a previous study in which a G-C base pair was inserted at position 1030-1124, at the base of h42, which connects to the GAC (Sergiev et al 2005). This mutation was predicted to lengthen and rotate h42, moving the GAC to a ''downward'' position.…”

Selenocysteine Insertion Sequence (SECIS)-binding Protein 2 Alters Conformational Dynamics of Residues Involved in tRNA Accommodation in 80 S Ribosomes

“…Helix 89 Modifications in Presence of SBP2-Previous chemical probing studies have correlated a conformational change in the SRL with the selective binding of EF-G over EF-Tu in pretranslocation state ribosomes (17). Additionally, movement of the GAC has also been implicated in EF binding specificity (18). In our analysis, there were no differences in the reactivity of residues in the GAC or the SRL in the presence of SBP2 (data not shown).…”

Pokeweed antiviral protein depurinates the sarcin/ricin loop of the rRNA prior to binding of aminoacyl-tRNA to the ribosomal A-site

“…occupancy status of the peptidyl-transferase center is allosterically transmitted to the factor-binding site, consisting of the GTPase-associated center and the sarcin/ricin loop. For example, structural probing experiments revealed a defined chain of nucleotides connecting the P-loop of the 23S rRNA to the elongation factor binding site, supporting the hypothesis that the occupancy status of the P-site determines the conformation of the movable GTPaseassociated center relative to the sarcin/ricin loop, to either enhance or inhibit EF-G binding (Sergiev et al 2005). In addition, extensive mutational analysis of the ribosomal protein L3 indicated that amino acid changes displaying the more pronounced mutant phenotypes were those in close proximity to functional regions of the 25S, the peptidyltransferase A-site and the sarcin/ricin loop.…”

“…This observation is relevant to the in vivo effects of PAP, given that eukaryotic cells contain excess amounts of eEF1A relative to eEF-2 and that eEF1A is not a limiting factor for protein synthesis (Slobin 1980). Recent structural analysis of the ribosome also showed that change in position of the GTPase-associated center relative to the sarcin/ricin loop altered the function of EF-G but not that of EF-Tu, suggesting that the structural requirements for activity of each elongation factor also differ (Sergiev et al 2005). Therefore, even though both factors bind the sarcin/ricin loop, the conformation of the loop and its depurination status may have differing effects on each factor.…”