V Ramakrishnan

Mrc Harwell, Oxford, England, United Kingdom

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Publications (122)1812.47 Total impact

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    ABSTRACT: In bacteria, disassembly of the ribosome at the end of translation is facilitated by an essential protein factor termed ribosome recycling factor (RRF), which works in concert with elongation factor G. Here we describe the crystal structure of the Thermus thermophilus RRF bound to a 70S ribosomal complex containing a stop codon in the A site, a transfer RNA anticodon stem-loop in the P site and tRNA(fMet) in the E site. The work demonstrates that structures of translation factors bound to 70S ribosomes can be determined at reasonably high resolution. Contrary to earlier reports, we did not observe any RRF-induced changes in bridges connecting the two subunits. This suggests that such changes are not a direct requirement for or consequence of RRF binding but possibly arise from the subsequent stabilization of a hybrid state of the ribosome.
    Nature Structural & Molecular Biology 09/2007; 14(8):733-7. DOI:10.1038/nsmb1282 · 11.63 Impact Factor
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    ABSTRACT: One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.
    Nature Structural & Molecular Biology 07/2007; 14(6):498-502. DOI:10.1038/nsmb1242 · 11.63 Impact Factor
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    ABSTRACT: During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem-loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASL(CCCG) and ASL(ACCC) (5'-3' nomenclature) form unpredicted anticodon-codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASL(ACGA) bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon-codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.
    RNA 07/2007; 13(6):817-23. DOI:10.1261/rna.367307 · 4.62 Impact Factor
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    ABSTRACT: Initiation of translation is the process by which initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site. In eukaryotes, one of the first steps involves the binding of two small factors, eIF1 and eIF1A, to the small (40S) ribosomal subunit. This facilitates tRNA binding, allows scanning of mRNA, and maintains fidelity of start codon recognition. Using cryo-EM, we have obtained 3D reconstructions of 40S bound to both eIF1 and eIF1A, and with each factor alone. These structures reveal that together, eIF1 and eIF1A stabilize a conformational change that opens the mRNA binding channel. Biochemical data reveal that both factors accelerate the rate of ternary complex (eIF2*GTP*Met-tRNA(i)(Met)) binding to 40S but only eIF1A stabilizes this interaction. Our results suggest that eIF1 and eIF1A promote an open, scanning-competent preinitiation complex that closes upon start codon recognition and eIF1 release to stabilize ternary complex binding and clamp down on mRNA.
    Molecular Cell 05/2007; 26(1):41-50. DOI:10.1016/j.molcel.2007.03.018 · 14.46 Impact Factor
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    ABSTRACT: 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.
    Science 10/2006; 313(5795):1935-42. DOI:10.1126/science.1131127 · 31.48 Impact Factor
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    ABSTRACT: During protein synthesis, translational release factors catalyze the release of the polypeptide chain when a stop codon on the mRNA reaches the A site of the ribosome. The detailed mechanism of this process is currently unknown. We present here the crystal structures of the ribosome from Thermus thermophilus with RF1 and RF2 bound to their cognate stop codons, at resolutions of 5.9 Angstrom and 6.7 Angstrom, respectively. The structures reveal details of interactions of the factors with the ribosome and mRNA, including elements previously implicated in decoding and peptide release. They also shed light on conformational changes both in the factors and in the ribosome during termination. Differences seen in the interaction of RF1 and RF2 with the L11 region of the ribosome allow us to rationalize previous biochemical data. Finally, this work demonstrates the feasibility of crystallizing ribosomes with bound factors at a defined state along the translational pathway.
    Cell 01/2006; 123(7):1255-66. DOI:10.1016/j.cell.2005.09.039 · 33.12 Impact Factor
  • Tina Daviter, Frank V Murphy, V Ramakrishnan
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    ABSTRACT: Decoding of the genetic message involves both the ribosome and its tRNA substrates. Most studies on the details of this process have focused on the role of the ribosome. In their Perspective, Daviter et al. discuss new work (Cochella and Green) that shows how the properties of tRNA influence its own selection.
    Science 06/2005; 308(5725):1123-4. DOI:10.1126/science.1113415 · 31.48 Impact Factor
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    James M Ogle, V Ramakrishnan
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    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. DOI:10.1146/annurev.biochem.74.061903.155440 · 26.53 Impact Factor
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    ABSTRACT: The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.
    Nature Structural & Molecular Biology 01/2005; 11(12):1186-91. DOI:10.1038/nsmb861 · 11.63 Impact Factor
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    Frank V Murphy, V Ramakrishnan
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    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. DOI:10.1038/nsmb866 · 11.63 Impact Factor
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    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. DOI:10.1107/S0907444903017669 · 7.23 Impact Factor
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    James M Ogle, Andrew P Carter, V Ramakrishnan
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    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. DOI:10.1016/S0968-0004(03)00066-5 · 13.52 Impact Factor
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    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. DOI:10.1126/science.1081798 · 31.48 Impact Factor
  • Ditlev E Brodersen, V Ramakrishnan
    Nature Structural Biology 03/2003; 10(2):78-80. DOI:10.1038/nsb0203-78
  • M. Valle, R. Gillet, S. Kaur, A. Henne, V. Ramakrishnan, J. Frank
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    ABSTRACT: Visualizing tmRNA Entry into a Stalled Ribosome
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    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. DOI:10.1016/S0092-8674(02)01086-3 · 33.12 Impact Factor
  • Biochemistry 04/2002; 32(47). DOI:10.1021/bi00210a033 · 3.19 Impact Factor
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    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. DOI:10.1006/jmbi.2001.5359 · 3.96 Impact Factor
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    V Ramakrishnan
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    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. DOI:10.1016/S0092-8674(02)00619-0 · 33.12 Impact Factor
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    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. DOI:10.1006/jmbi.2001.4778 · 3.96 Impact Factor

Publication Stats

12k Citations
1,812.47 Total Impact Points

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Institutions

  • 2001–2014
    • Mrc Harwell
      Oxford, England, United Kingdom
  • 2001–2013
    • Medical Research Council (UK)
      Londinium, England, United Kingdom
  • 2002–2011
    • University of Cambridge
      • MRC Laboratory of Molecular Biology
      Cambridge, ENG, United Kingdom
  • 2010
    • MRC Cognition and Brain Sciences Unit
      Cambridge, England, United Kingdom
  • 2009
    • The Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom
  • 1996–2000
    • University of Utah
      • Department of Biochemistry
      Salt Lake City, Utah, United States
  • 1987–2000
    • Brookhaven National Laboratory
      • Biology Department
      New York City, NY, 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
  • 1990
    • University of Adelaide
      Tarndarnya, South Australia, Australia
  • 1988
    • Yale University
      • Department of Molecular Biophysics and Biochemistry
      New Haven, Connecticut, United States
  • 1984
    • University of New Haven
      New Haven, Connecticut, United States