Download or read book Escherichia Coli 30S Ribosomal Subunit Assembly written by Jennifer Anne Maki and published by . This book was released on 2004 with total page 206 pages. Available in PDF, EPUB and Kindle. Book excerpt: The prokaryotic ribosome is a 2.5 MDa particle comprised of two asymmetric subunits, the large (50S) and small (30S) subunits. The large subunit contains two RNAs (5S and 23S) in addition to thirty-four proteins. The small subunit consists of one RNA (16S rRNA) and twenty-one proteins. Although the crystal structure has been solved, much remains to be revealed concerning the assembly of this macromolecular structure. Our laboratory is focused on the assembly of the small subunit. In vitro assembly of this structure was achieved in the late 1960's and early 1970's. At low temperature when 16S rRNA and all of the small subunit proteins are incubated together, only a subset of the proteins are able to associate with the RNA and a particle termed Reconstitution Intermediate (RI, 2IS) results. When RI particles are heat treated, a conformational rearrangement occurs and RI* (26S) particles result that are capable of complete assembly with the remainder of the small subunit proteins, even at low temperature, to form functional 30S subunits. In vitro 30S subunit assembly requires long incubation periods, high ionic strength, and heat treatment. In light of these strict requirements, we hypothesized that assembly factors must exist in vivo to facilitate this crucial assembly process, making it accurate and efficient. We have identified the DnaK chaperone system as one such factor. The purified DnaK chaperone system is sufficient to facilitate in vitro 30S subunit assembly at low temperature, forming 30S particles that co-sediment, have the same protein complement, bind tRNA, and participate in polyphenylalanine synthesis like 30S subunits. Additionally, the association behavior of the DnaK chaperone system components with pre-30S particles in vitro was observed and found to be very similar to their association with substrate in their well-characterized protein folding role. Lastly, it was determined that DnaK binds small subunit components in vivo, including pre-processed 16S rRNA. This is the first evidence clearly demonstrating a direct link between the DnaK chaperone system and the assembly of ribosomes in E. coli, and the first instance in which an extra-ribosomal assembly factor has been shown to facilitate 30S subunit assembly in vitro.

Download or read book Analysis of E Coli 30S Ribosomal Subunit Assembly Kinetics by Quantitative Mass Spectrometry written by Anne Elizabeth Bunner and published by . This book was released on 2009 with total page 316 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribosome biogenesis is an essential process in all living cells. The E. coli 30S ribosomal subunit self-assembles in vitro in a cooperative manner, with the RNA-binding of some proteins enabled by the prior binding of others under equilibrium conditions. We have developed a pulse-chase monitored by quantitative mass spectrometry (PC/QMS) method for monitoring in vitro 30S ribosome assembly kinetics. This approach utilizes liquid-chromatography coupled mass spectrometry (LC/MS) analysis of stable-isotope labeled peptides, and quantitation is achieved using an isotope distribution fitting approach. This method was applied to the study of cooperativity and the rate-limiting steps in 30S ribosomal subunit assembly. The experiments revealed that kinetic cooperativity does not always align with thermodynamic cooperativity, and that the protein S19 is a secondary organizer of the 3' domain. In addition, this method was used to investigate the effect of assembly co-factors Era, RimM, and RimP on in vitro assembly kinetics. Each individual assembly factor caused significant and specific kinetic changes in ribosome assembly, which provide important clues to the mechanism of these proteins.

Download or read book Assembly of the 30S Ribosomal Subunit written by Joel F. Grondek and published by . This book was released on 2004 with total page 72 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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 over 100Å 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, polyspecific 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.

Download or read book Assembly of the 30S Ribosomal Subunit written by Indumathi Jagannathan and published by . This book was released on 2004 with total page 286 pages. Available in PDF, EPUB and Kindle. Book excerpt: The aim of this study is to dissect molecular events that occur during the assembly of 30S subunit of the E. coli ribosome. The 30S ribosomal subunit is made up of 16S ribosomal RNA (rRNA) and 21 proteins (S1-S21). Crystal structure of the 30S subunit has answered longstanding questions on the three-dimensional organization of its constituents; however, this view does not expose the nature of interesting cooperative movements and conformational changes that occur during the formation of this macromolecular complex. Hence, biochemical and genetic efforts are required to understand these rearrangements. In this study, we have employed directed hydroxyl radical probing and used one of the ribosomal proteins (r-proteins) S15 as the probe to identify conformational changes that occur in 16S rRNA during different stages of assembly. S15 is one of the proteins that directly interact with the 16S rRNA and it governs the binding of four other proteins during 30S subunit formation. S15 has been converted into a probe by substituting cysteines at unique positions of the protein and attaching Fe(II) to the cysteines to form Fe(II) tethered S15 proteins (Fe(II)-S15). Hydroxyl radicals can be generated from these tethered sites by Fenton chemistry. The "directed hydroxyl radicals so produced cleave RNA elements that are in proximity to the tethered sites and these cleavage sites are mapped by primer extension. Using the recombinant in vitro reconstitution system and this directed hydroxyl radical probing approach, we have studied the folding of 16S rRNA proximal to S15 during the course of 30S subunit assembly. These studies have revealed protein-dependent conformational changes that occur in RNA environment of S15. Our work suggests that binding of r-proteins can result in changes that are quite remote from their primary binding site and that assembly of different domains can influence one another.

Download or read book 30S Ribosomal Subunit Assembly is a Target for Inhibition by Aminoglycoside Antibiotics in Escherichia Coli written by Roopal Manoj Mehta and published by . This book was released on 2002 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Antibacterial agents specific for the 50S ribosomal subunit not only inhibit translation but also prevent assembly of that subunit. I examined the 30S ribosomal subunit in growing Escherichia coli cells to see if antibiotics specific for that subunit also had a second inhibitory effect. I used the aminoglycoside antibiotics paromomycin and neomycin, which bind specifically to the 30S ribosomal subunit. Both antibiotics inhibited the growth rate, viable cell number, and protein synthesis. I used a 3H-uridine pulse and chase assay to examine the kinetics of ribosome subunit assembly in the presence and absence of each antibiotic. Analysis revealed a concentration dependent inhibition of 30S subunit formation in the presence of each antibiotic. Sucrose gradient profiles of cell lysates showed the accumulation of an intermediate 21S translational particle. Taken together this data gives the first demonstration that 30S ribosomal subunit inhibitors can also prevent assembly of the small subunit.

Download or read book The Structural Heterogeneity of the 30S Ribosomal Subunit from Escherichia Coli written by Paul Voynow and published by . This book was released on 1970 with total page 190 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Studies on the Anatomy of Ribosomes from Escherichia Coli written by Pallaiah Thammana and published by . This book was released on 1974 with total page 268 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Studies of Functional Domains in Ribosomal Proteins of the Escherichia Coli 30S Ribosomal Subunit written by Richard C. Conrad and published by . This book was released on 1987 with total page 464 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book The Assembly Landscape of the 30S Ribosomal Subunit Monitored with Mass Spectrometry written by Megan Wright Trevathan and published by . This book was released on 2004 with total page 626 pages. Available in PDF, EPUB and Kindle. Book excerpt: The architecture of the bacterial ribosome is now known at atomic resolution. A similarly detailed picture of how the rRNA and protein components assemble into this structure is still being drawn. In this work, a new method is developed to probe the mechanism of assembly of the E. coli 30S ribosomal subunit. The method, which measures RNA-protein binding kinetics in vitro with isotopic labeling and quantitative protein mass spectrometry, monitors simultaneously the binding kinetics of individual proteins to the assembling 30S subunit and thus reports on the kinetics of assembling the many parts of the structure. The method reveals that the proteins generally bind in single kinetic phases. The events limiting protein binding are a mix of bimolecular (rRNA-protein binding) and unimolecular (folding) transitions. In no case is folding dramatically slower than binding. 30S assembly is understood to be limited by an energy-dependent conformational change of a single intermediate. Data from this study indicate that assembling 30S subunits populate multiple intermediates; three are proposed, each of which corresponds to a particular region of the 30S structure. One follows initial assembly of the 5 ' domain and some assembly in the central domain. The second follows further 5' domain assembly. The third follows assembly of the central domain. Much of the 3 ' domain, along with the mRNA decoding site at the junction of the three domains, assembles after the last intermediate. It appears that no single transition is particularly energy-dependent; rather, the many steps of assembly have similar activation energies. Mg2+ is needed in high concentrations (at least 10 mM) for complete, efficient 30S assembly. Here it is shown that high [Mg 2+ ] is required for the later steps, in which the 3' domain and mRNA decoding site are assembled. The earlier steps of assembly, however, are slowed down by high [Mg2+ ]. A study of the folding of another RNA, the Tetrahymena group I ribozyme, shows that unfolding of structure can play a large role in RNA folding. Similarly, it appears that Mg2+ slows down the early steps of 30S assembly by preventing unfolding of intermediate structures and possibly by stabilizing misfolded intermediates.

Download or read book Site specific 3 terminal Deletions in Escherichia Coli 16S RRNA and the Effect on Assembly of 30S Ribosomal Subunits written by Fazilah Abdul-Latif and published by . This book was released on 1984 with total page 132 pages. Available in PDF, EPUB and Kindle. Book excerpt: The ribosome is a central component of the protein synthetic apparatus. It is a macromolecular complex of protein and RNA. Although much progress has been made in understanding the functional role of the proteins in this particle, little is known of the functional role which the RNA plays. Naturally occurring primary structural mutations in rRNAs have not been reported. The work described here focused on developing methodology for generating site-specific deletions in rRNA directly to explore the functional properties of the RNA. The 3'-terminus of E. coli small subunit 16S rRNA was chosen as the site to be deleted. This region of the RNA is believed to be important in the initiation of protein synthesis and could be essential for proper assembly of ribosomes. The deletions were achieved by synthesizing a DNA molecule of 10 bases in length which was complementary to the 3'-end of 16S rRNA. The DNA was hybridized to the RNA and then the RNA component of the hybrid was specifically digested with a combination of RNase H isolated from E. coli and calf thymus. This produced an equal mixture of "16S" RNAs missing either 10 or 8 terminal nucleotides. This RNA was shown to be functional in in vitro reconstitution studies indicating that this zone of the RNA is not essential for ribosome assembly.

Download or read book Role of GTPases in Assembly of the Bacterial 30S Subunit written by Aida Razi and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "My PhD thesis aims to improve our understanding of the assembly process of the bacterial ribosome and the overarching goal of my research is to identify steps in this pathway that can be targeted with new molecular probes. Ribosome assembly in actively growing Escherichia Coli involves multiple events including synthesis of 54 ribosomal-proteins and their binding to ribosomal RNAs in a hierarchical manner. Many of these events are assisted by a number of accessory proteins known as assembly factors, making the process extremely fast and efficient. My PhD thesis contributes new knowledge on the precise mechanism by which YjeQ and Era assist the ribosome assembly process. Recent work indicates that both factors assist the late stages of the 30S subunit assembly. To reveal the precise role of YjeQ in the context of the mature 30S subunit, we solved the structure of the 30S subunit in complex with YjeQ by cryo-electron microscopy (Cryo-EM). Our data suggested that in addition to work as a maturation factor, YjeQ also functions as a checkpoint protein testing the proofreading ability of the ribosomal subunit prior to the particle's release into the pool of actively translating ribosomes. This work provides the first example of a bacterial assembly factor that tests a specific translation mechanism of the 30S subunit. To investigate the role of Era, we used quantitative mass spectrometry (qMS), microscale thermophoresis (MST) and cryo-EM techniques. We first determined using cryo-EM the structures of the assembly intermediate that accumulated in E. coli upon depletion of Era at 3.8Å resolution. In addition, we also solved the structure of the mature 30S subunit in complex with Era. We found that Era-depleted cells accumulated three assembly intermediates at different stages of maturation ranging from early stage of maturation to the late stage of 30S subunit with missing density both in the central and 3' minor domains of the 30S subunit. Densities for ribosomal proteins (r-proteins) bS21, bS1, uS3 and uS2 bound to the central domain were also missing from the late stage of 30S subunit. The structure of the 30S subunit in complex with Era demonstrated that binding of this factor reverts the mature subunit to an immature state lacking density for the motifs integrating the decoding center. These results suggest that Era facilitates the proper folding of the platform region of the 30S subunit. In addition, Era may work as a placeholder for some of the late ribosomal proteins and prevent premature association of the 30S subunit with the 50S subunit. Finally, we tested whether YjeQ and Era exert their function in conjunction rather than independently. To this end, we obtained the cryo-EM structure of the 30S subunit in complex with Era and YjeQ at 3.5Å resolution. However, we couldn't observe any density for YjeQ in this structure. Moreover, we demonstrated Era binds to the cavity between head and platform and reverts the structure of the 30S subunit to its immature state. These results suggest that Era and YjeQ works subsequently during assembly. Era binds first to the assembling subunit and assists the folding of the platform region at the same time that it prevents the binding of YjeQ. Once Era is released, YjeQ binds assisting the maturation of the decoding center and testing the fidelity of the subunit before releasing it to the pool of fully active ribosomes. Overall, this thesis provides novel insights into the function of two important GTPases dedicated to the assembly process of the bacterial ribosome." --

Download or read book Translation Initiation Reconstituted Systems and Biophysical Methods written by and published by Academic Press. This book was released on 2007-10-23 with total page 512 pages. Available in PDF, EPUB and Kindle. Book excerpt: For over fifty years the Methods in Enzymology series has been the critically aclaimed laboratory standard and one of the most respected publications in the field of biochemistry. The highly relevant material makes it an essential publication for researchers in all fields of life and related sciences. This volume, the second of three on the topic of Translation Initiation includes articles written by leaders in the field.

Download or read book Dissecting the Relationships Between RNA Structure and Function in E Coli Small Ribosomal Subunits written by Zhili Xu and published by . This book was released on 2010 with total page 402 pages. Available in PDF, EPUB and Kindle. Book excerpt: "Ribonucleic acids (RNAs) perform widely different biological functions ranging from regulation of gene expression to catalysis. Their functions are often tightly correlated with their structure. To explore the relationship between RNA structure and function, Escherichia coli (E. coli) small ribosomal subunit was used as a model system and its biogenesis was investigated using two different approaches. Firstly, we studied a RNA modification system which involves dimethylation of two universally conserved adjacent adenosines in the ultimate helix of the small subunit ribosomal RNAs (rRNA) and the KsgA (E. coli) methyltransferase family, enzyme responsible for these two dimethylations, which also appears to be universally conserved. We localized the position of KsgA relative to 30S subunits using directed hydroxyl radical probing and demonstrated that binding of KsgA and translation initiation factor 3 (IF3) are generally mutually exclusive. Our work strongly suggested that this modification system plays a critical role in differentiating fully assembled, functional subunits from those that are still involved in the biogenesis pathway. Secondly, we devised a modification interference approach to investigate nucleotides within 16S rRNA that are potentially important for the assembly of functional 30S subunits. We found that the majority of these nucleotides are located in the head and interdomain junction of the 30S subunit indicating that the different domains of the 30S subunit assemble differentially and that the assembly of the 3' domain and interdomain junction are more restricted than assembly of the other domains. Further in vivo investigation of individual residues identified in vitro delineated the importance of these residues in ribosome biogenesis. Overall, this dissertation has demonstrated the impact of 16S rRNA structural changes on biogenesis of the small ribosomal subunit and has enabled deeper understanding of ribosome assembly."--Leaves v-vi.

Download or read book Protein Synthesis and Ribosome Structure written by Knud H. Nierhaus and published by John Wiley & Sons. This book was released on 2004-10-15 with total page 608 pages. Available in PDF, EPUB and Kindle. Book excerpt: Knud Nierhaus, who has studied the ribosome for more than 30 years, has assembled here the combined efforts of several scientific disciplines into a uniform picture of the largest enzyme complex found in living cells, finally resolving many decades-old questions in molecular biology. In so doing he considers virtually all aspects of ribosome structure and function -- from the molecular mechanism of different ribosomal ribozyme activities to their selective inhibition by antibiotics, from assembly of the core particle to the regulation of ribosome component synthesis. The result is a premier resource for anyone with an interest in ribosomal protein synthesis, whether in the context of molecular biology, biotechnology, pharmacology or molecular medicine.

Download or read book Structural Studies of the Ribosome of Escherichia Coli written by Leonard Charles Lutter and published by . This book was released on 1973 with total page 344 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Role of Long Range Interactions in Assembly of the Small Subunit of Escherichia Coli Ribosome written by Deepika S. Calidas and published by . This book was released on 2012 with total page 136 pages. Available in PDF, EPUB and Kindle. Book excerpt: "The function of the small subunit (SSU) of the ribosome of Escherichia coli is dependent on dynamic interactions at the intersection of its four domains; namely, the body, platform, head and penultimate stem. The in vitro assembly of each individual domain from its corresponding structural element in 16S ribosomal RNA (rRNA), i.e., the 5', central, 3' major and minor domains and associated ribosomal proteins (r-proteins) has been extensively researched. Less is understood of the long range interactions that occur during assembly as different domains co-assemble, both in vitro and in vivo. Our first approach was to use directed probing from the S8 r-protein as a monitor of SSU assembly. We found that assembly of the neck, a functionally significant region between the head and platform is dependent on assembly of the body. Furthermore, S8 binds two distinct binding sites in 16S rRNA separated by several hundred nucleotides, and the appropriate architecture of the later transcribed region is dependent upon incorporation of r-proteins to the earlier transcribed region. Elements of the body domain, including the 5' terminus do not assume their appropriate conformation except upon assembly of the entire domain. Also, we found that S12 could influence the architecture of the 5' terminus, leading us to examine the role of S12 in 30S subunit assembly, both in vitro and in vivo. S12 possesses a non-canonically structured extension that extends from the solvent surface to the intersubunit surface of the SSU, contacting multiple domains. An almost complete truncation of the extension was unable to support growth, while partial truncations of more than 6 amino acids exhibited growth defects. Truncation of half or all of the extension also resulted in reduced activity of SSUs assembled in vitro. The architecture of ribonucleoprotein complexes assembled with truncated proteins is also altered. The work presented in this thesis elucidates influence of widely separated elements of the SSU on each other during assembly"--Page v.

Download or read book Ribosomes and Protein Synthesis written by Gary Spedding and published by IRL Press. This book was released on 1990 with total page 352 pages. Available in PDF, EPUB and Kindle. Book excerpt: A practical and self-contained introduction to methods of researching the structure and function of the ribosome in light of the increasing recognition of the potential capability of RNA molecules to act as molecular catalysts. Also describes protein synthesis and cell-free synthesizing systems. Annotation copyrighted by Book News, Inc., Portland, OR