Escherichia Coli 30S Ribosomal Subunit Assembly PDF Download
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Author: Jennifer Anne Maki
Publisher:
ISBN:
Category :
Languages : en
Pages : 206
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Book Description
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.
Author: Jennifer Anne Maki
Publisher:
ISBN:
Category :
Languages : en
Pages : 206
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Book Description
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.
Author: Anne Elizabeth Bunner
Publisher:
ISBN: 9781109683738
Category : Mass spectrometry
Languages : en
Pages : 316
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Book Description
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.
Author: Joel F. Grondek
Publisher:
ISBN:
Category :
Languages : en
Pages : 72
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Book Description
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.
Author: Indumathi Jagannathan
Publisher:
ISBN:
Category :
Languages : en
Pages : 286
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Book Description
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.
Author: Roopal Manoj Mehta
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
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.
Author: Paul Voynow
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 190
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Author: Pallaiah Thammana
Publisher:
ISBN:
Category :
Languages : en
Pages : 268
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Author: Richard C. Conrad
Publisher:
ISBN:
Category :
Languages : en
Pages : 464
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Author: Megan Wright Trevathan
Publisher:
ISBN:
Category : Chemical kinetics
Languages : en
Pages : 626
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Book Description
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.
Author: Fazilah Abdul-Latif
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 132
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Book Description
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.
