Comprehensive genetic selection revealed essential bases in the peptidyl-transferase center

Abstract

During protein synthesis, the ribosome catalyzes peptide-bond formation. Biochemical and structural studies revealed that conserved nucleotides in the peptidyl-transferase center (PTC) and its proximity may play a key role in peptide-bond formation; the exact mechanism involved remains unclear. To more precisely define the functional importance of the highly conserved residues, we used a systematic genetic method, which we named SSER (systematic selection of functional sequences by enforced replacement), that allowed us to identify essential nucleotides for ribosomal function from randomized rRNA libraries in Escherichia coli cells. These libraries were constructed by complete randomization of the critical regions in and around the PTC. The selected variants contained natural rRNA sequences from other organisms and organelles as well as unnatural functional sequences; hence providing insights into the functional roles played by these essential bases and suggesting how the universal catalytic mechanism of peptide-bond formation could evolve in all living organisms. Our results highlight essential bases and interactions, which are shaping the PTC architecture and guiding the motions of the tRNA terminus from the A to the P site, found to be crucial not only for the formation of the peptide bond but also for nascent chain elongation.nucleotide essentiality ͉ protein biosynthesis ͉ symmetrical region R ibosomes are universally conserved ribonucleoproteins that translate the genetic information contained in mRNAs into proteins. The large (50S) ribosomal subunit catalyzes peptide-bond formation at the peptidyl-transferase center (PTC) between aminoacyl-tRNA (aa-tRNA) bound to the A site and peptidyl-tRNA (pep-tRNA) at the P site. In the crystal structures of 50S subunits of Haloarcula marismortui, H50S (1, 2), Deinococcus radiodurans, D50S (3, 4), and 70S ribosomes (5, 6), the PTC is composed solely of 23S rRNA and, hence, acts as a ribozyme, consistent with biochemical findings using deproteinized 50S subunit (7-10) for the formation of a single peptide bond.The PTC provides the frame for peptide-bond formation (11, 12) and plays a critical role in tRNA and nascent chain release (13-17), and the global ribosomal architecture is crucial for substrate positioning (18,19). Consistently, the hypothesis, based on structures of H50S complexed with minimum substrates, that the PTC acts as a general acid-base catalyst (2, 20-22), was contradicted by various mutagenesis and biochemical studies (12,(23)(24)(25)(26)(27)(28). The main catalytic contribution of the ribosomes, substrates positioning at proper orientation (4,28,29), is achieved by remote interactions, accompanied by symmetrical base-pairing of C75 of both tRNAs with G2553 (Escherichia coli numbering throughout) and G2251 (Fig. 4A, which is published as supporting information on the PNAS web site), respectively (2,4,31). The distinction between the rates of peptide-bond formation by full-length tRNAs and minimal substrates is also consistent with the essential role ...

Mutations of highly conserved bases in the peptidyltransferase center induce compensatory rearrangements in yeast ribosomes

“…In a prior study using E. coli, the U2492, C2556, and C2573 (yeast U2861, C2925, and C2942) accommodation gate bases were identified among other functionally important nucleotides by systematic selection of functional sequences by enforced replacement (SSER) (Sato et al 2006). In a similar study, C2556 (yeast C2925) was also identified and proposed to be actively involved in translation (Blanchard and Puglisi 2001;Yassin and Mankin 2007).…”

Ribosome's mode of function: myths, facts and recent results

“…Consistently, the quantum mechanical calculations indicated that the transition state (TS) for this reaction is formed during the rotatory motion, and is stabilized by the interactions of the rotating 3 end with the ribosome components of the rear wall [30]. The location of the computed TS is similar to that observed crystallographically for mimics of the TS in the large ribosomal subunit from another source, namely H50S [31], and the significance of the interactions between the rotating moiety and the PTC walls was verified by comprehensive mutagenesis analysis [32].…”

Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

“…Hence, the ribosomal architecture and its mobility provide all structural elements enabling the ribosome to function as an amino acid polymerase, including the formation of two symmetrical universal base pairs between the tRNAs and the PTC Agmon et al 2005), a prerequisite for substrate-mediated acceleration (Weinger & Strobel 2006) and for the direction of the nascent protein into the exit tunnel. Importantly, all nucleotides involved in this rotatory motion have been classified as essential by a comprehensive genetic selection analysis (Sato et al 2006). Furthermore, the rotatory motion positions the proximal 2 0 -hydroxyl of P-site tRNA A76 in the same position and orientation found in crystals of the entire ribosome with mRNA and tRNAs, as determined independently in two laboratories (Korostelev et al 2006;Selmer et al 2006), and allows for chemical catalysis of peptide bond formation by A76 of the P-site tRNA (Weinger & Strobel 2006).…”

“…The structure of the large ribosomal subunit from D. radiodurans (D50S) in complexes with a substrate analogue mimicking the A-site tRNA part interacting with the large subunit, called ASM, advanced the comprehension of peptide bond formation by showing that ribosomes position their substrates in stereochemistry suitable for peptide bond formation, thus providing the machinery for peptide bond formation and tRNA translocation Agmon et al 2005). Furthermore, the ribosomal architecture, which facilitates positional catalysis of peptide bond formation, promotes substrate-mediated chemical acceleration in accord with the requirement of full-length tRNAs for rapid and smooth peptide bond formation, observed by various methods, including the use of chemical (Weinger et al 2004;Brunelle et al 2006;Weinger & Strobel 2006), mutagenesis (Sato et al 2006), computational (Sharma et al 2005;Gindulyte et al 2006;Trobro & Aqvist 2006) and kinetic procedures (Beringer et al 2005;Wohlgemuth et al 2006;Rodnina et al 2007). The current consensus view is consistent with ribosomal positional catalysis that allows for chemical catalysis by its P-site tRNA substrate.…”

“…At the A-and P-sites, the tRNA anticodon loops interact with the mRNA on the small subunit, and the acceptor stem with the aminoacylated or peptidylated 3 0 end is located on the large subunit. A-to P-site tRNA translocation comprises two highly correlated motions: sideways shift and a ribosomal navigated rotatory motion (figure 2; Agmon et al 2003Agmon et al , 2005Agmon et al , 2006Agmon et al , 2009Bashan et al 2003;Sato et al 2006;Bashan & Yonath 2008b), during which peptide bonds are being formed (Gindulyte et al 2006). This process also involves the translocation of the tRNA 3 0 end from the A-to the P-site, the detachment of the P-site tRNA from the growing polypeptide chain, the passage of the deacylated tRNA molecule to the E-site and its subsequent release.…”