The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
V Ramakrishnan - One of the best experts on this subject based on the ideXlab platform.
-
crystal structure of a bioactive pactamycin analog bound to the 30S Ribosomal Subunit
Journal of Molecular Biology, 2013Co-Authors: David S Tourigny, Israel S Fernandez, Ann C. Kelley, Ramkrishna Reddy Vakiti, Amit Chattopadhyay, Stephane Dorich, Stephen Hanessian, V RamakrishnanAbstract:Biosynthetically and chemically derived analogs of the antibiotic pactamycin and de-6-methylsalicylyl (MSA)-pactamycin have attracted recent interest as potential antiprotozoal and antitumor drugs. Here, we report a 3.1-A crystal structure of de-6-MSA-pactamycin bound to its target site on the Thermus thermophilus 30S Ribosomal Subunit. Although de-6-MSA-pactamycin lacks the MSA moiety, it shares the same binding site as pactamycin and induces a displacement of nucleic acid template bound at the E-site of the 30S. The structure highlights unique interactions between this pactamycin analog and the ribosome, which paves the way for therapeutic development of related compounds.
-
modification of 16s Ribosomal rna by the ksga methyltransferase restructures the 30S Subunit to optimize ribosome function
RNA, 2010Co-Authors: Hasan Demirci, A C Kelley, V Ramakrishnan, F V Murphy, Riccardo Belardinelli, Steven T Gregory, Albert E Dahlberg, G JoglAbstract:All organisms incorporate post-transcriptional modifications into Ribosomal RNA, influencing ribosome assembly and function in ways that are poorly understood. The most highly conserved modification is the dimethylation of two adenosines near the 39 end of the small Subunit rRNA. Lack of these methylations due to deficiency in the KsgA methyltransferase stimulates translational errors during both the initiation and elongation phases of protein synthesis and confers resistance to the antibiotic kasugamycin. Here, we present the X-ray crystal structure of the Thermus thermophilus 30S Ribosomal Subunit lacking these dimethylations. Our data indicate that the KsgA-directed methylations facilitate structural rearrangements in order to establish a functionally optimum Subunit conformation during the final stages of ribosome assembly.
-
mechanism for expanding the decoding capacity of transfer rnas by modification of uridines
Nature Structural & Molecular Biology, 2007Co-Authors: A Weixlbaumer, F V Murphy, Agnieszka Dziergowska, Andrzej Malkiewicz, Franck A P Vendeix, Paul F Agris, V RamakrishnanAbstract: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 GU 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.
-
phasing the 30S Ribosomal Subunit structure
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Ditlev E Brodersen, William M Clemons, Brian T Wimberly, Andrew P Carter, V RamakrishnanAbstract: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.
-
crystal structure of an initiation factor bound to the 30S Ribosomal Subunit
Science, 2001Co-Authors: Andrew P Carter, William M Clemons, Brian T Wimberly, Ditlev E Brodersen, Robert J Morganwarren, Thomas Hartsch, V RamakrishnanAbstract: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.
Gloria M. Culver - One of the best experts on this subject based on the ideXlab platform.
-
assembly of the 30S Ribosomal Subunit
EcoSal Plus, 2008Co-Authors: Gloria M. Culver, Narayanaswamy KirthiAbstract:Protein synthesis involves nearly a third of the total molecules in a typical bacterial cell. Within the cell, protein synthesis is performed by the ribosomes, and research over several decades has investigated Ribosomal formation, structure, and function. This review provides an overview of the current understanding of the assembly of the Escherichia coli 30S Ribosomal Subunit. The E. coli 30S Subunit contains one rRNA molecule (16S) and 21 Ribosomal proteins (r-proteins; S1 to S21). The formation of functional Subunits can occur as a self-assembly process in vitro; i.e., all the information required for the formation of active ribosomes resides in the primary sequences of the r-proteins and rRNAs. In vitro reconstitution of functional 30S Subunits is carried out by using a mixture of TP30, individually purified natural or recombinant r-proteins, and natural 16S rRNA. Chemical probing and primer extension analysis have been used extensively to monitor changes in the reactivities of nucleotides in 16S rRNA during the in vitro reconstitution of 30S Subunits. The potential roles for r-proteins in 30S Subunit assembly were determined by omitting single proteins in reconstitution experiments. The RNPs resulting from single protein omissions were examined in terms of their composition and function to determine the roles of the absent proteins. Recent developments in understanding the structure of the 30S Subunit have led to speculation about roles for some of the r-proteins in assembly. The crystal structures of the 30S Subunit ( 1 , 2 ) and the 70S ribosome ( 3 ) reveal details of the r-protein and rRNA interactions.
-
assembly of the 30S Ribosomal Subunit positioning Ribosomal protein s13 in the s7 assembly branch
RNA, 2004Co-Authors: Joel F Grondek, Gloria M. CulverAbstract:Studies of Escherichia coli 30S Ribosomal Subunit assembly have revealed a hierarchical and cooperative association of Ribosomal proteins with 16S Ribosomal RNA; these results have been used to compile an in vitro 30S Subunit assembly map. In single protein addition and omission studies, Ribosomal protein S13 was shown to be dependent on the prior association of Ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S Subunit > 100 A away from S20, which resides near the bottom of the body of the 30S Subunit. All of the proteins that reside in the head of the 30S Subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S Subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3' major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S Subunit assembly map where its position is in accordance with much biochemical and structural data.
-
mapping structural differences between 30S Ribosomal Subunit assembly intermediates
Nature Structural & Molecular Biology, 2004Co-Authors: Kristi L Holmes, Gloria M. CulverAbstract:Under appropriate conditions, functional Escherichia coli 30S Ribosomal Subunits assemble in vitro from purified components. However, at low temperatures, assembly stalls, producing an intermediate (RI) that sediments at 21S and is composed of 16S Ribosomal RNA (rRNA) and a subset of Ribosomal proteins (r-proteins). Incubation of RI at elevated temperatures produces a particle, RI*, of similar composition but different sedimentation coefficient (26S). Once formed, RI* rapidly associates with the remaining r-proteins to produce mature 30S Subunits. To understand the nature of this transition from RI to RI*, changes in the reactivity of 16S rRNA between these two states were monitored by chemical modification and primer extension analysis. Evaluation of this data using structural and biochemical information reveals that many changes are r-protein–dependent and some are clustered in functional regions, suggesting that this transition is an important step in functional 30S Subunit formation.
-
assembly of the central domain of the 30S Ribosomal Subunit roles for the primary binding Ribosomal proteins s15 and s8
Journal of Molecular Biology, 2003Co-Authors: Indu Jagannathan, Gloria M. CulverAbstract:Assembly of the 30S Ribosomal Subunit occurs in a highly ordered and sequential manner. The ordered addition of Ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S Ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S Subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S Subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.
-
Assembly of the 30S Ribosomal Subunit.
Biopolymers, 2003Co-Authors: Gloria M. CulverAbstract:Ribosomes are large macromolecular complexes responsible for cellular protein synthesis. The smallest known cytoplasmic ribosome is found in prokaryotic cells; these ribosomes are about 2.5 MDa and contain more than 4000 nucleotides of RNA and greater than 50 proteins. These components are distributed into two asymmetric Subunits. Recent advances in structural studies of ribosomes and Ribosomal Subunits have revealed intimate details of the interactions within fully assembled particles. In contrast, many details of how these massive ribonucleoprotein complexes assemble remain elusive. The goal of this review is to discuss some crucial aspects of 30S Ribosomal Subunit assembly.
William M Clemons - One of the best experts on this subject based on the ideXlab platform.
-
phasing the 30S Ribosomal Subunit structure
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Ditlev E Brodersen, William M Clemons, Brian T Wimberly, Andrew P Carter, V RamakrishnanAbstract: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.
-
recognition of cognate transfer rna by the 30S Ribosomal Subunit
Science, 2001Co-Authors: James M Ogle, William M Clemons, Ditlev E Oderse, Michael J Tarry, Andrew P Carte, V RamakrishnaAbstract: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. During protein synthesis, the ribosome catalyzes the sequential addition of amino acids to a growing polypeptide chain, using mRNA as a template and aminoacylated tRNAs (aatRNAs) as substrates. Correct base pairing between the three bases of the codon on mRNA and those of the anticodon of the cognate aatRNA dictates the sequence of the polypeptide
-
crystal structure of an initiation factor bound to the 30S Ribosomal Subunit
Science, 2001Co-Authors: Andrew P Carter, William M Clemons, Brian T Wimberly, Ditlev E Brodersen, Robert J Morganwarren, Thomas Hartsch, V RamakrishnanAbstract: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.
-
the structural basis for the action of the antibiotics tetracycline pactamycin and hygromycin b on the 30S Ribosomal Subunit
Cell, 2000Co-Authors: D E Brodersen, William M Clemons, Brian T Wimberly, Andrew P Carter, Robert J Morganwarren, V RamakrishnanAbstract: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.
-
functional insights from the structure of the 30S Ribosomal Subunit and its interactions with antibiotics
Nature, 2000Co-Authors: Andrew P Carter, William M Clemons, Brian T Wimberly, Ditlev E Brodersen, Robert J Morganwarren, V RamakrishnanAbstract: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.
James R Williamson - One of the best experts on this subject based on the ideXlab platform.
-
kinetic cooperativity in escherichia coli 30S Ribosomal Subunit reconstitution reveals additional complexity in the assembly landscape
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Anne E Bunner, Andrea H Beck, James R WilliamsonAbstract:The Escherichia coli 30S Ribosomal Subunit self-assembles in vitro in a hierarchical manner, with the RNA binding by proteins enabled by the prior binding of others under equilibrium conditions. Early 16S rRNA binding proteins also bind faster than late-binding proteins, but the specific causes for the slow binding of late proteins remain unclear. Previously, a pulse-chase monitored by quantitative mass spectrometry method was developed for monitoring 30S Subunit assembly kinetics, and here a modified experimental scheme was used to probe kinetic cooperativity by including a step where subsets of Ribosomal proteins bind and initiate assembly prior to the pulse-chase kinetics. In this work, 30S Ribosomal Subunit kinetic reconstitution experiments revealed that thermodynamic dependency does not always correlate with kinetic cooperativity. Some folding transitions that cause subsequent protein binding to be more energetically favorable do not result in faster protein binding. Although 3′ domain primary protein S7 is required for RNA binding by both proteins S9 and S19, prior binding of S7 accelerates the binding of S9, but not S19, indicating there is an additional mechanistic step required for S19 to bind. Such data on kinetic cooperativity and the presence of multiphasic assembly kinetics reveal complexity in the assembly landscape that was previously hidden.
-
a complex assembly landscape for the 30S Ribosomal Subunit
Annual Review of Biophysics, 2009Co-Authors: Michael T Sykes, James R WilliamsonAbstract:The ribosome is a complex macromolecular machine responsible for protein synthesis in the cell. It consists of two Subunits, each of which contains both RNA and protein components. Ribosome assembly is subject to intricate regulatory control and is aided by a multitude of assembly factors in vivo, but can also be carried out in vitro. The details of the assembly process remain unknown even in the face of atomic structures of the entire ribosome and after more than three decades of research. Some of the earliest research on ribosome assembly produced the Nomura assembly map of the small Subunit, revealing a hierarchy of protein binding dependencies for the 20 proteins involved and suggesting the possibility of a single intermediate. Recent work using a combination of RNA footprinting and pulse-chase quantitative mass spectrometry paints a picture of small Subunit assembly as a dynamic and varied landscape, with sequential and hierarchical RNA folding and protein binding events finally converging on complete Subunits. Proteins generally lock tightly into place in a 5' to 3' direction along the Ribosomal RNA, stabilizing transient RNA conformations, while RNA folding and the early stages of protein binding are initiated from multiple locations along the length of the RNA.
-
an assembly landscape for the 30S Ribosomal Subunit
Nature, 2005Co-Authors: Gary Siuzdak, Megan W T Talkington, James R WilliamsonAbstract:Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, mRNA processing, translation, DNA replication, and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S Subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, due to the challenges of monitoring the association of many components simultaneously. We have developed a new method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 Ribosomal proteins with 16S rRNA during formation of the functional particle. These data represent the first detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we have found that local transformations throughout the assembling Subunit have similar but distinct activation energies. This observation shows that the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to a view in which the assembly of the complex traverses a landscape dotted with a variety of local conformational transitions.
-
assembly of the 30S Ribosomal Subunit
Quarterly Reviews of Biophysics, 2005Co-Authors: James R WilliamsonAbstract:The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway.
Brian T Wimberly - One of the best experts on this subject based on the ideXlab platform.
-
phasing the 30S Ribosomal Subunit structure
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Ditlev E Brodersen, William M Clemons, Brian T Wimberly, Andrew P Carter, V RamakrishnanAbstract: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.
-
crystal structure of an initiation factor bound to the 30S Ribosomal Subunit
Science, 2001Co-Authors: Andrew P Carter, William M Clemons, Brian T Wimberly, Ditlev E Brodersen, Robert J Morganwarren, Thomas Hartsch, V RamakrishnanAbstract: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.
-
the structural basis for the action of the antibiotics tetracycline pactamycin and hygromycin b on the 30S Ribosomal Subunit
Cell, 2000Co-Authors: D E Brodersen, William M Clemons, Brian T Wimberly, Andrew P Carter, Robert J Morganwarren, V RamakrishnanAbstract: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.
-
functional insights from the structure of the 30S Ribosomal Subunit and its interactions with antibiotics
Nature, 2000Co-Authors: Andrew P Carter, William M Clemons, Brian T Wimberly, Ditlev E Brodersen, Robert J Morganwarren, V RamakrishnanAbstract: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.
-
structure of the 30S Ribosomal Subunit
Nature, 2000Co-Authors: Brian T Wimberly, William M Clemons, Andrew P Carter, Ditlev E Brodersen, Robert J Morganwarren, Thomas Hartsch, Clemens Vonrhein, V RamakrishnanAbstract: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.
