Mutational Analysis of Two Highly Conserved UGG Sequences of 23 S rRNA from Escherichia coli

Abstract

The 23 S-type rRNA contains two phylogenetically conserved UGG sequences, which have the potential to bind the universal CCA-3-ends of tRNAs at the ribosomal peptidyltransferase center by base pairing. The first two positions, UG, of these sequences at the helix-loop 80 (U2249G2250) and helix-loop 90 (⌿2580G2581) and some related nucleotides were tested by site-directed mutagenesis for their involvement in ribosomal function, i.e. peptidyltransferase. The plasmid-derived mutated 23 S rRNA comprised about 50% of the total 23 S rRNA. None of the single mutations caused an assembly defect, and all 50 S subunits carrying an altered 23 S rRNA could freely exchange with the pools of 70S ribosomes and polysomes. The mutations at the helix-loop 80 region hardly affected bacterial growth. However, mutations at the helix 90 caused severe growth effects and severely impaired the in vitro protein synthesis, showing that this 23 S rRNA region is of high importance for ribosomal function.The central enzymatic activity of ribosomes is the formation of peptide bonds. The corresponding peptidyltransferase (PTF) 1 center is located on the large ribosomal subunit (1, 2). Reconstitution analyses have identified the ribosomal proteins L2, L3, and L4, and the 23 S rRNA as PTF candidates in Escherichia coli ribosomes (3-5). A complex derived from the large subunit of Thermus aquaticus ribosomes consisting of 23 S rRNA and only 3-8 proteins had significant PTF activity (6, 7). This observation underscores the possible involvement of 23 S rRNA in this activity.An impressive wealth of data points to a distinct feature of the secondary structure map of 23 S rRNA, the so-called "peptidyltransferase ring" of domain V (8, 9) as a component at or near the PTF center. The PTF ring comprises about 40 nucleotides and represents a cluster of universally conserved nucleotides (10, 11). It is satisfying that a highly divergent array of methods correspondingly identifies the same region of 23 S rRNA, methods such as cross-linking studies with substrates of the PTF center (12-15), mutations of the 23 S rRNA gene conferring resistance against inhibitors of the PTF activity (for review, see Refs. 16 and 17), and protection studies (18 -20).Some studies aimed to identify nucleotides at the PTF center. The nucleotides G2252 and G2253, which are at the helixloop 80, were protected against kethoxal by the 3Ј-terminal CCA of P-site bound tRNA (19). Mutation of G2252 generated a dominant lethal phenotype (21). A double mutant having both Gs altered was lethal and showed a reduced activity of peptide bond formation by approximately 50% (22). A recent analysis presented evidence that G2252 is involved in canonical base pairing with C74 at the acceptor end of tRNA, thus obviously playing a role in the binding of the donor substrate at the P site region of the PTF center (23).Another study applied a random mutagenesis of the "Southern half" of the PTF ring (residues 2493-2606). With an elegant screening procedure, 21 mutations of 18 positions were found. Mutatio...

Conserved Nucleotides of 23 S rRNA Located at the Ribosomal Peptidyltransferase Center

“…Mutants of the first sequence did not influence ribosomal assembly and bacterial growth but caused some defects in in vitro poly(U) translation. Those of the second sequence are characterized by a seemingly normal assembly but with severe defects in peptide synthesis (1). The ⌿GG2582 sequence is located at the "peptidyltransferase ring" (see Fig.…”

“…1). Here we analyze the peptidyltransferase (PTF) 1 activity in various systems and the translocation reaction. All mutant ribosomes were able to translocate.…”

“…The first two positions of both sequences were mutated, and the effects on ribosomal assembly and functions were analyzed (1). Mutants of the first sequence did not influence ribosomal assembly and bacterial growth but caused some defects in in vitro poly(U) translation.…”

Functional Importance of the 3′-Terminal Adenosine of tRNA in Ribosomal Translation

“…A-to P-site Translocation-To assay the effect of G 76 on the translocation of tRNA from the ribosomal A-site to the P-site, the ribosomal Pand E-sites were filled with deacylated tRNA CCA in the presence of poly(GUA) by incubating 20 l of mix R, containing 5 pmol of 70 S ribosomes, 25 pmol of poly(GUA) 44 (25). Translocation was promoted by the addition of variable amounts of EF-G (0 -2.5 pmol) and 1 mM GTP (final concentration) and incubating for either 10 min at 37°C or 2 h on ice (final volume, 35 l).…”

Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors

“…In the last 20 years, molecular reengineering of ribosomal RNA (rRNA) has been widely used for understanding the mechanisms of protein synthesis and translation regulation Dahlberg 1993, 1996;Gregory et al 1994;Spahn et al 1996;Bocchetta et al 1998;O'Connor et al 2001; Thompson et al 2001;Cochella and Green 2004;Sergiev et al 2005;Hirabayashi et al 2006;Walker et al 2008;Persaud et al 2010;Burakovsky et al 2011). The method usually requires introducing a mutated region in the ribosomal operon inserted into a plasmid under a strong inducible promoter.…”