V Ramakrishnan

University of Cambridge, Cambridge, ENG, United Kingdom

Are you V Ramakrishnan?

Claim your profile

Publications (115)1622.95 Total impact

  • Source
    James M Ogle, V Ramakrishnan
    [Show abstract] [Hide abstract]
    ABSTRACT: The underlying basis for the accuracy of protein synthesis has been the subject of over four decades of investigation. Recent biochemical and structural data make it possible to understand at least in outline the structural basis for tRNA selection, in which codon recognition by cognate tRNA results in the hydrolysis of GTP by EF-Tu over 75 A away. The ribosome recognizes the geometry of codon-anticodon base pairing at the first two positions but monitors the third, or wobble position, less stringently. Part of the additional binding energy of cognate tRNA is used to induce conformational changes in the ribosome that stabilize a transition state for GTP hydrolysis by EF-Tu and subsequently result in accelerated accommodation of tRNA into the peptidyl transferase center. The transition state for GTP hydrolysis is characterized, among other things, by a distorted tRNA. This picture explains a large body of data on the effect of antibiotics and mutations on translational fidelity. However, many fundamental questions remain, such as the mechanism of activation of GTP hydrolysis by EF-Tu, and the relationship between decoding and frameshifting.
    Annual Review of Biochemistry 02/2005; 74:129-77. · 27.68 Impact Factor
  • Source
    Frank V Murphy, V Ramakrishnan
    [Show abstract] [Hide abstract]
    ABSTRACT: Here we report the crystal structures of I.C and I.A wobble base pairs in the context of the ribosomal decoding center, clearly showing that the I.A base pair is of an I(anti).A(anti) conformation, as predicted by Crick. Additionally, the structures enable the observation of changes in the anticodon to allow purine-purine base pairing, the 'widest' base pair geometry allowed in the wobble position.
    Nature Structural & Molecular Biology 01/2005; 11(12):1251-2. · 11.90 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The methods involved in determining the 850 kDa structure of the 30S ribosomal subunit from Thermus thermophilus were in many ways identical to those that are generally used in standard protein crystallography. This paper reviews and analyses the methods that can be used in phasing such large structures and shows that the anomalous signal collected from heavy-atom compounds bound to the RNA is both necessary and sufficient for ab initio structure determination at high resolution. In addition, measures to counter problems with non-isomorphism and radiation decay are described.
    Acta Crystallographica Section D Biological Crystallography 12/2003; 59(Pt 11):2044-50. · 14.10 Impact Factor
  • Source
    James M Ogle, Andrew P Carter, V Ramakrishnan
    [Show abstract] [Hide abstract]
    ABSTRACT: During the decoding process, tRNA selection by the ribosome is far more accurate than expected from codon-anticodon pairing. Antibiotics such as streptomycin and paromomycin have long been known to increase the error rate of translation, and many mutations that increase or lower accuracy have been characterized. Recent crystal structures show that the specific recognition of base-pairing geometry leads to a closure of the domains of the small subunit around cognate tRNA. This domain closure is likely to trigger subsequent steps in tRNA selection. Many antibiotics and mutations act by making the domain closure more or less favourable. In conjunction with recent cryoelectron microscopy structures of the ribosome, a comprehensive structural understanding of the decoding process is beginning to emerge.
    Trends in Biochemical Sciences 06/2003; 28(5):259-66. · 13.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at approximately 13 to 15 angstroms of the entry of tmRNA into the ribosome. The structure reveals how tmRNA could move through the ribosome despite its complicated topology and also suggests roles for proteins S1 and SmpB in the function of tmRNA.
    Science 05/2003; 300(5616):127-30. · 31.20 Impact Factor
  • Ditlev E Brodersen, V Ramakrishnan
    Nature Structural Biology 03/2003; 10(2):78-80.
  • M. Valle, R. Gillet, S. Kaur, A. Henne, V. Ramakrishnan, J. Frank
    [Show abstract] [Hide abstract]
    ABSTRACT: Visualizing tmRNA Entry into a Stalled Ribosome
    Science. 01/2003; 300:127.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A structural and mechanistic explanation for the selection of tRNAs by the ribosome has been elusive. Here, we report crystal structures of the 30S ribosomal subunit with codon and near-cognate tRNA anticodon stem loops bound at the decoding center and compare affinities of equivalent complexes in solution. In ribosomal interactions with near-cognate tRNA, deviation from Watson-Crick geometry results in uncompensated desolvation of hydrogen-bonding partners at the codon-anticodon minor groove. As a result, the transition to a closed form of the 30S induced by cognate tRNA is unfavorable for near-cognate tRNA unless paromomycin induces part of the rearrangement. We conclude that stabilization of a closed 30S conformation is required for tRNA selection, and thereby structurally rationalize much previous data on translational fidelity.
    Cell 12/2002; 111(5):721-32. · 31.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure. Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA. Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later. We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit.
    Journal of Molecular Biology 03/2002; 316(3):725-68. · 3.91 Impact Factor
  • Source
    V Ramakrishnan
    [Show abstract] [Hide abstract]
    ABSTRACT: The publication of crystal structures of the 50S and 30S ribosomal subunits and the intact 70S ribosome is revolutionizing our understanding of protein synthesis. This review is an attempt to correlate the structures with biochemical and genetic data to identify the gaps and limits in our current knowledge of the mechanisms involved in translation.
    Cell 03/2002; 108(4):557-72. · 31.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 A resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 A resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.
    Journal of Molecular Biology 08/2001; 310(4):827-43. · 3.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or "wobble," position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs.
    Science 06/2001; 292(5518):897-902. · 31.03 Impact Factor
  • Source
    V Ramakrishnan, P B Moore
    [Show abstract] [Hide abstract]
    ABSTRACT: Last year, atomic structures of the 50S ribosomal subunit from Haloarcula marismortui and of the 30S ribosomal subunit from Thermus thermophilus were published. A year before that, a 7.8 A resolution electron density map of the 70S ribosome from T. thermophilus appeared. This information is revolutionizing our understanding of protein synthesis.
    Current Opinion in Structural Biology 05/2001; 11(2):144-54. · 8.74 Impact Factor
  • Cold Spring Harbor Symposia on Quantitative Biology 02/2001; 66:17-32.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit.
    Science 02/2001; 291(5503):498-501. · 31.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We have used the recently determined atomic structure of the 30S ribosomal subunit to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B. The antibiotics bind to discrete sites on the 30S subunit in a manner consistent with much but not all biochemical data. For each of these antibiotics, interactions with the 30S subunit suggest a mechanism for its effects on ribosome function.
    Cell 01/2001; 103(7):1143-54. · 31.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.
    Nature 10/2000; 407(6802):327-39. · 38.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.
    Nature 09/2000; 407(6802):340-348. · 38.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: X-ray crystallography has recently yielded much-improved electron-density maps of the bacterial ribosome and its two subunits and many structural details of bacterial ribosome subunits are now being resolved. One approach to complement the structures and elucidate the details of rRNA and protein packing is to determine structures of individual protein components and model these into existing intermediate resolution electron density. We have determined the solution structure of the ribosomal protein S16 from Thermus thermophilus. S16 is a mixed alpha/beta protein with a novel folding scaffold based on a five-stranded antiparallel/parallel beta sheet. Three large loops, which are partially disordered, extend from the sheet and two alpha helices are packed against its concave surface. Calculations of surface electrostatic potentials show a large continuous area of positive electrostatic potential and smaller areas of negative potential. S16 was modeled into a 5.5 A electron-density map of the T. thermophilus 30S ribosomal subunit. The location and orientation of S16 in a narrow crevice formed by helix 21 and several other unassigned rRNA helices is consistent with electron density corresponding to the shape of S16, hydroxyl radical protection data, and the electrostatic surface potential of S16. Two protein neighbors to S16 are S4 and S20, which facilitate binding of S16 to the 30S subunit. Overall, this work exemplifies the benefits of combining high-resolution nuclear magnetic resonance (NMR) structures of individual components with low-resolution X-ray maps to elucidate structures of large complexes.
    Structure 09/2000; 8(8):875-82. · 5.99 Impact Factor
  • Source
    F Shu, V Ramakrishnan, B P Schoenborn
    [Show abstract] [Hide abstract]
    ABSTRACT: Although hydrogens comprise half of the atoms in a protein molecule and are of great importance chemically and structurally, direct visualization of them by using crystallography is difficult. Neutron crystallography is capable of directly revealing the position of hydrogens, but its use on unlabeled samples faces certain technical difficulties: the large incoherent scattering of hydrogen results in background scattering that greatly reduces the signal to noise of the experiment. Moreover, whereas the scattering lengths of C, N, and O are positive, that of hydrogen is negative and about half the magnitude. This results in density for hydrogens being half as strong and close to the threshold of detection at 2.0-A resolution. Also, because of its opposite sign, there is a partial cancellation of the hydrogen density with that from neighboring atoms, which can lead to ambiguities in interpretation at medium resolution. These difficulties can be overcome by the use of deuterated protein, and we present here a neutron structure of fully deuterated myoglobin. The structure reveals a wealth of chemical information about the molecule, including the geometry of hydrogen bonding, states of protonation of histidines, and the location and geometry of water molecules at the surface of the protein. The structure also should be of broader interest because it will serve as a benchmark for molecular dynamics and energy minimization calculations and for comparison with NMR studies.
    Proceedings of the National Academy of Sciences 05/2000; 97(8):3872-7. · 9.81 Impact Factor

Publication Stats

9k Citations
1,622.95 Total Impact Points

Top Journals

Institutions

  • 2002–2013
    • University of Cambridge
      • MRC Laboratory of Molecular Biology
      Cambridge, ENG, United Kingdom
  • 2001–2013
    • Medical Research Council (UK)
      Londinium, England, United Kingdom
  • 2012
    • Uppsala University
      • Department of Cell and Molecular Biology
      Uppsala, Uppsala, Sweden
  • 2011
    • McGill University
      Montréal, Quebec, Canada
  • 2009
    • The Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom
  • 1996–2000
    • University of Utah
      • Department of Biochemistry
      Salt Lake City, UT, United States
    • Stony Brook University
      Stony Brook, New York, United States
  • 1987–2000
    • Brookhaven National Laboratory
      • Biology Department
      New York City, NY, United States
  • 1998
    • St. Jude Children's Research Hospital
      • Department of Structural Biology
      Memphis, TN, United States
  • 1997
    • University of Texas at Austin
      • Department of Chemistry and Biochemistry
      Texas City, TX, United States
  • 1993–1996
    • Duke University Medical Center
      Durham, North Carolina, United States
  • 1982–1988
    • Yale University
      • Department of Molecular Biophysics and Biochemistry
      New Haven, Connecticut, United States
  • 1982–1984
    • University of New Haven
      New Haven, Connecticut, United States