Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. In addition, although past structures of the 50S subunit have found no ordered proteins at the PTC, biochemical evidence suggests that specific proteins are capable of interacting with the 3′ ends of tRNA ligands. Here we present structures, at 3.6-Å and 3.5-Å resolution respectively, of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl-transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. They also reveal interactions between the ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer

Moment inversion problem for piecewise D-finite functions

We consider the problem of exact reconstruction of univariate functions with jump discontinuities at unknown positions from their moments. These functions are assumed to satisfy an a priori unknown linear homogeneous differential equation with polynomial coefficients on each continuity interval. Therefore, they may be specified by a finite amount of information. This reconstruction problem has practical importance in Signal Processing and other applications. It is somewhat of a ``folklore'' that the sequence of the moments of such ``piecewise D-finite''functions satisfies a linear recurrence relation of bounded order and degree. We derive this recurrence relation explicitly. It turns out that the coefficients of the differential operator which annihilates every piece of the function, as well as the locations of the discontinuities, appear in this recurrence in a precisely controlled manner. This leads to the formulation of a generic algorithm for reconstructing a piecewise D-finite function from its moments. We investigate the conditions for solvability of the resulting linear systems in the general case, as well as analyze a few particular examples. We provide results of numerical simulations for several types of signals, which test the sensitivity of the proposed algorithm to noise

Insights into Translational Termination from the Structure of RF2 Bound to the Ribosome

The termination of protein synthesis occurs through the specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site transfer RNA. Here we present, at a resolution of 3.5 angstroms, the crystal structure of RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon; it also suggests a model for the role of a universally conserved GGQ motif in the catalysis of peptide release

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. In addition, although past structures of the 50S subunit have found no ordered proteins at the PTC, biochemical evidence suggests that specific proteins are capable of interacting with the 3′ ends of tRNA ligands. Here we present structures, at 3.6-Å and 3.5-Å resolution respectively, of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl-transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. They also reveal interactions between the ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer

Mechanism of eIF6 release from the nascent 60S ribosomal subunit.

SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS-EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias.Supported by a Federation of European Biochemical Societies Long term Fellowship (to FW), Specialist Programme from Bloodwise [12048] (AJW), the Medical Research Council [MC_U105161083] (AJW) and [U105115237] (RRK), Wellcome Trust strategic award to the Cambridge Institute for Medal Research [100140], Tesni Parry Trust (AJW), Ted’s Gang (AJW) and the Cambridge NIHR Biomedical Research Centre.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nsmb.311

Structural basis of RNA polymerase III transcription initiation.

RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4-4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation

Structure of the 70S ribosome complexed with mRNA and tRNA

The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation

Structural biology:Pivotal findings for a transcription machine

Crystal structures of the complete RNA Polymerase I complex are now revealed. These structures link opening and closing of the DNA binding cleft of this enzyme to control of transcription

Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release

We report the crystal structure of release factor 2 bound to ribosome with an aminoacyl tRNA substrate analog at the ribosomal P site, at 3.1 Å resolution. The structure shows that upon stop-codon recognition, the universally conserved GGQ motif packs tightly into the peptidyl transferase center. Nucleotide A2602 of 23S rRNA, implicated in peptide release, packs with the GGQ motif in release factor 2. The ribose of A76 of the peptidyl-tRNA adopts the C2′-endo conformation, and the 2′ hydroxyl of A76 is within hydrogen-bond distance of the 2′ hydroxyl of A2451. The structure suggests how a catalytic water can be coordinated in the peptidyl transferase center and, together with previous biochemical and computational data, suggests a model for how the ester bond between the peptidyl tRNA and the nascent peptide is hydrolyzed