Modulation of Translational Fidelity by Small Subunit Ribosoma RNA from Escherichia Coli PDF Download
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Author: Fazilah Abdul-Latif
Publisher:
ISBN:
Category : Proteins
Languages : en
Pages : 198
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Book Description
Ribosomes are ribonucleoprotein particles that are essential for the process of protein synthesis in all living systems. Escherichia coli, eubacterial organisms, have ribosomes which display a 70S sedimentation coefficient. They are formed by the association of two ribonucleoprotein subunits which sediment at 50S and 30S. The 30S subunit is a construct made of 21 different proteins and 16S ribosomal RNA. The 3'-terminus of this rRNA was the focus of the research recounted in this dissertation. This tract of 10 nucleotides in rRNA is absolutely conserved in eubacterial organisms and is asserted to be crucial for proper initiation of protein synthesis in eubacteria. Here I report the results of experiments done with 16S rRNA missing a part of the conserved 3'-terminus. These experiments were undertaken to provide a refinement of our understanding of the requirement for and function of this conserved segment of 16S rRNA. The deletions were made directly within mature 16S rRNA using RNase H and a 10 nucleotide synthetic DNA complementary to the 3'-terminus of 16S rRNA. The synthetic DNA was hybridized to 16S rRNA and then treated with RNase H. RNase H will only attack RNA when it is basepaired with DNA. This permits site-directed mutagenisis on the mature RNA. This deletion strategy efficiently yielded a 3'-terminal nucleotide deletion in E. coli 16S rRNA. This 3'-terminal deletion did not impair in vitro 30S subunit assembly. Therefore, the conservation of the sequence is not necessary for ribosome assembly. To investigate the functional properties of the modified particles an in vitro protein translation system primed with a natural mRNA was employed. The mRNA was viral MS2 messenger. Modified particles did translate the MS2 mRNA but the fidelity with which the translation occurred was diminished.
Author: Fazilah Abdul-Latif
Publisher:
ISBN:
Category : Proteins
Languages : en
Pages : 198
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Book Description
Ribosomes are ribonucleoprotein particles that are essential for the process of protein synthesis in all living systems. Escherichia coli, eubacterial organisms, have ribosomes which display a 70S sedimentation coefficient. They are formed by the association of two ribonucleoprotein subunits which sediment at 50S and 30S. The 30S subunit is a construct made of 21 different proteins and 16S ribosomal RNA. The 3'-terminus of this rRNA was the focus of the research recounted in this dissertation. This tract of 10 nucleotides in rRNA is absolutely conserved in eubacterial organisms and is asserted to be crucial for proper initiation of protein synthesis in eubacteria. Here I report the results of experiments done with 16S rRNA missing a part of the conserved 3'-terminus. These experiments were undertaken to provide a refinement of our understanding of the requirement for and function of this conserved segment of 16S rRNA. The deletions were made directly within mature 16S rRNA using RNase H and a 10 nucleotide synthetic DNA complementary to the 3'-terminus of 16S rRNA. The synthetic DNA was hybridized to 16S rRNA and then treated with RNase H. RNase H will only attack RNA when it is basepaired with DNA. This permits site-directed mutagenisis on the mature RNA. This deletion strategy efficiently yielded a 3'-terminal nucleotide deletion in E. coli 16S rRNA. This 3'-terminal deletion did not impair in vitro 30S subunit assembly. Therefore, the conservation of the sequence is not necessary for ribosome assembly. To investigate the functional properties of the modified particles an in vitro protein translation system primed with a natural mRNA was employed. The mRNA was viral MS2 messenger. Modified particles did translate the MS2 mRNA but the fidelity with which the translation occurred was diminished.
Author: Fazilah Abdul Latif
Publisher:
ISBN:
Category :
Languages : en
Pages : 30
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Book Description
The ribosome is a central component of the protein synthetic apparatus. Although progress has been made in characterizing the functional role of many of the ribosomal proteins, the properties of ribosomal RNA and its role in robosomal structure and function are not well understood. To investigate the working properties of the highly conserved 3'-end of 16S rRNA, a site-specific deletion was made directly within the 16S rRNA molecule. The terminal delection did not impair in vitro 30S subunit assembly, but the praticles produced lost translational fidelity in an vitro translation system primed with natural mRNA. [Authors' abstract].
Author: Piotr Lagod
Publisher:
ISBN:
Category :
Languages : en
Pages : 68
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Book Description
Ribosomes translate messenger RNA (mRNA) three nucleotides at a time until translation is terminated at a stop codon. However, during all translation, frameshifting can occur, leading to the formation of proteins with amino acid sequences that differ from the in-frame product. Spontaneous frameshifting can be harmful to an organism. For instance, antibiotics such as streptomycin inhibit bacterial growth by increasing misreading and frameshifting. However, programmed translational frameshifting (which can induce high levels of frameshifting) can be used in some instances to control the ratio of specific proteins (as seen with the dnaX gene) or to increase the density of genomic information. This study explored the effects of endogenous small molecules on the IS3-frameshift-motif that is found in the transposase genes of many mobile elements. Using a cell-free protein synthesis system and a luminescent frameshift reporter, it was discovered that the addition of a small molecule extract derived from E. coli significantly decreased frameshifting, suggesting that it contains molecules that can alter translational fidelity. These experiments also revealed that the addition of the translation inhibitor chloramphenicol to translation assays at sub-inhibitory concentrations, reduced frameshift efficiency. During the studies, the role of the stability of luminescent protein reporters on the reported frameshifting levels was also explored, which is omitted in many studies. Finally, a method was developed that allows for the isolation of molecules that weakly associate with ribosomes, which opens the door for more detailed investigations of chemicals that alter translational fidelity. In conclusion, these studies provide new insight on the potential modulation of translational frameshifting by endogenous small molecules, and they set the stage to reveal the important players in this important biochemical process.
Author: Sue Jinks Robertson
Publisher:
ISBN:
Category : Proteins
Languages : en
Pages : 464
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Book Description
Author: Young Sook Yoo
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 168
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Book Description
Ribosomes are intricate macromolecular complexes which are a major element of the protein biosynthetic machinery in all life forms. In Escherichia coli they contain about 50 distinct proteins and 3 ribosomal RNAs. The small 30S ribosomal subunit in E. coli incorporates 21 proteins and a 16S rRNA. The 16S rRNA associated with this subunit was the focus of the investigations described in this dissertation. In order to explore the functional properties of this rRNA an in vitro procedure was developed to generate site-specific internal deletions in the RNA. The C-1400 region of the 16S rRNA was selected for manipulation because the sequence in this zone of the molecule has been shown to be intrinsically universal in all sequenced small subunit rRNAs. Through the use of synthetic DNA, RNase H, and RNA ligase, a four-nucleotide deletion between positions 1400 and 1405 was constructed. The manipulated RNA was tested for competency in in vitro ribosome reconstitution experiments and yielded particles which manifest a sedimentation coefficient comparable to normal 30S subunits. Therefore, this portion of the conserved sequence did not emerge to be a ribosome assembly imperative and must fulfill an essential function during translation.
Author: Patrick Nikita Allen
Publisher:
ISBN:
Category : RNA.
Languages : en
Pages : 250
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Author: Aishwarya Devaraj
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
Abstract: Protein synthesis occurs in ribosomes, megadalton RNA-protein machines that use aminoacyl-tRNA (aa-tRNA) molecules to translate messenger RNA (mRNA) with high fidelity. During translation elongation, the ribosome orchestrates 3 major events: decoding, peptidyl transfer and translocation. The process of proteins synthesis is also one of the major targets of antibiotics and hence understanding the basics of ribosome function should provide insight for the development of novel drugs. Genomes are maintained and expressed with remarkable fidelity and the accuracy of each process involved represents a compromise that optimizes the evolutionary fitness of the organism. The process of translation elongation is a complex one, and therefore there are potentially many ways the process can go awry. Chapter 1 introduces translation elongation errors and discusses the differences between missense, nonsense and frameshift errors. Mutations in the ribosome and other translation factors that affect the fidelity of translation elongation are also discussed. Chapter 2 is focused on the ribosomal exit (E) site and its role in maintaining the translational reading frame. It has been proposed that a critical role for the E site is in maintenance of translational reading frame, dependent on codon-anticodon pairing (191). Though several studies support the idea that codon-anticodon interaction in the E site contributes to frame maintenance (167), direct in vivo evidence for this hypothesis has been scant. In chapter 2, we investigated this fundamental question and found that the E site helps to maintain the reading frame, but does not contribute to the accuracy of decoding, as has been suggested (chapter 2, 204). We also showed that the mutation of the 30S E site does not inhibit EF-G-catalyzed translocation, in sharp contrast to the effects of mutations in 50S E site. These data provided evidence that the function of the E site in translocation is largely confined to the 50S subunit. One of the earliest identified examples of translational frameshifting occurs in the prfB gene of E. coli, encoding the peptide release factor 2 (RF2). While the genetic studies have identified the determinants of prfB programmed frameshifting and their relative importance, how these determinants act to promote frameshifting has remained unclear. In chapter 3, we compared ribosomal complexes with various spacer lengths between the SD sequence and P codon. We found that a close juxtaposition of the SD-ASD helix and P codon strongly destabilized P-site tRNA but had little or no effect on RF2-dependent termination or EF-Tu-dependent decoding. These data suggested that the intragenic SD of prfB destabilizes pairing of peptidyl-tRNALeu to the zero-frame CUU and promotes directional movement of the mRNA template with respect to the bound tRNA. In chapter 4, we have isolated 16S rRNA mutations that could suppress a +1 frameshift mutation in E. coli. In one of the screens (where the slippery sequence in the frameshift window had a stop codon), 31 independent mutations were identified and mapped to four different positions, of which C1054U was isolated 28 times. The C1054U mutation has also been isolated previously as a nonsense suppressor. Purine substitutions at this position also increased UGA readthrough and miscoding. While the C1054U mutation significantly increased nonsense readthrough and frameshift errors, the mutation had a hyperaccurate phenotype with respect to decoding (i.e., reduced misreading). Other substitutions at this position also had differential effects on the three reporters (missense, nonsense and frameshift). These interesting observations prompted us to characterize these A-site mutations as well as others in 16S rRNA (C1200U, G1491A and G299A) in vitro to get a better understanding of how the ribosome maintains its high fidelity (chapter 5). We investigated the effect of these mutations on RF2 function and found that all of the mutations tested had a defect in RF2-dependent termination. We directly tested the effect of these mutations on decoding by measuring the rate of GTP hydrolysis in both cognate and near-cognate mRNA. We found that all of the mutations tested (C1200U, G1491A, C1054U, C1054A, and G299A) had a substantial defect in initial selection, increasing the rate of GTP hydrolysis particularly on near-cognate mRNA. We also investigated the effect of these mutations on the stability of various tRNAs in the A site. Of the mutations analyzed, C1054U and G1491A seemed to differentially affect tRNA stability, suggesting that these mutations may stimulate GTP hydrolysis in a different way than the others.
Author: Corey M. Dambacher
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 366
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Book Description
The prokaryotic translational machinery is comprised of multiple components, including the ribosome, and aminoacyl tRNA synthetase enzymes. The roles of various ribosomal proteins have been studied for several decades, however many of these proteins are encoded by essential genes, making studies of ribosomal proteins very difficult. Many of the ribosomal proteins are involved in auto-regulatory networks, and direct assembly of the ribosome. Primary binding proteins nucleate assembly, while secondary and tertiary binding proteins can be required for passage from non-productive assembly intermediates to native assembly conformations. Studies of assembly have led to an increased understanding of the pathway in which this macromolecule is formed, but these data have been primarily obtained using in vitro reconstitution experiments. More recent studies have revealed a variety of intermediates in vivo that have not been seen in vitro. In vivo experiments designed to identify new intermediates are limited to over-expression analysis, as many of the essential ribosomal proteins when deleted, do not give rise to viable cells. Additionally, existing induction systems only allow for one to induce expression of target proteins, while to study the prokaryotic ribosome in the absence of individual ribosomal proteins, one would need to shut off expression in mid-growth phase. Here we report the generation of 41 E. coli ribosomal deletion strains that enable facile studies of all essential ribosomal proteins in this organism. To address dynamic repression and induction of these proteins, we have generated a bi-directional semi-oscillatory repression/induction switch (using an unnatural aminoacyl tRNA synthetase). Taken together, these results will allow investigators to access the entire ribosomal protein network for in vivo studies of regulation and ribosomal assembly.
Author: Gary Spedding
Publisher: IRL Press
ISBN:
Category : Science
Languages : en
Pages : 352
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Book Description
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
Author: Deepika S. Calidas
Publisher:
ISBN:
Category :
Languages : en
Pages : 136
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Book Description
"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.
