The Construction and Analysis of Mutant Small E. Coli Ribosomal Subunits with Inserts in the 16S RRNA PDF Download
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Author: Andrew Scheinman
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 288
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Book Description
Author: Andrew Scheinman
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 288
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Book Description
Author: William Roberts Staplin
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 136
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Author: Neha Gupta
Publisher:
ISBN:
Category :
Languages : en
Pages : 180
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Book Description
"Ribonucleoproteins (RNPs) perform diverse biological functions, from catalysis to regulation of gene expression. Ribosomes are complex RNPs that synthesize proteins in all living organisms. Ribosome assembly in bacteria has been studied using in vitro reconstitution experiments for over five decades. In vitro reconstitution is not truly representative of ribosome assembly as in vivo ribosome biogenesis requires transcription, processing and modification of 5000 nucleotides of ribosomal RNA (rRNA), and is aided by several auxiliary factors, which are generally not included in in vitro reconstitution. In vivo ribosome biogenesis in bacteria is poorly understood because it is a complex, efficient and asynchronous process with only a small pool of intermediates present in wild-type cells at any given time. This thesis presents novel techniques and findings on the assembly of ribosomal small subunit (SSU) in wild-type E. coli under optimal growth conditions. To study SSU assembly in E. coli, we developed RNP affinity purification techniques to isolate and characterize in vivo formed SSU intermediates. These approaches took advantage of the regions in precursor 16S rRNA (pre-16S rRNA, leader and trailer) that are components of the pre- rRNA and SSU intermediates but are absent in mature SSUs or ribosomes. An RNA affinity tag was inserted in pre-16S rRNA at different positions between various nucleolytic cleavage sites, allowing systematic purification of different intermediates and mapping of the assembly cascade. The first precursor of 16S rRNA (17S rRNA) is the major platform for SSU biogenesis in vivo. Structural probing demonstrated that these purified 17S rRNA containing SSU intermediates had diverse architectures representing early to late stages of SSU biogenesis. These intermediates are likely incapable of translation as the regions of 16S rRNA involved in translation showed altered structure compared to the corresponding regions in mature SSUs suggesting there are checkpoints that prevent immature subunits to enter the translation cycle. The three pre-SSUs exhibited differential association of ribosomal proteins and known auxiliary factors and thus revealed multiple pathways for these processes during SSU biogenesis. Several novel and putative auxiliary factors were also identified using proteomic analysis, and preliminary characterization had demonstrated their role in SSU biogenesis. Additionally, substrates for two 16S rRNA modification enzymes were partially characterized. Our results indicate that there are multiple pathways for different biogenesis processes and SSU assembly occurs largely on 17S rRNA. Final 17S rRNA processing events happen late in the biogenesis cascade and follow multiple pathways with independent 5ʹ end or 3ʹ end maturation of 17S rRNA. These findings allow the first integration of rRNA processing events with conformational changes, rRNA modification, r-proteins association and auxiliary factor action during ribosomal SSU biogenesis in wild-type bacteria"--Pages v-vi.
Author: K.H. Nierhaus
Publisher: Springer Science & Business Media
ISBN: 1461524075
Category : Science
Languages : en
Pages : 741
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Proceedings of an international conference held in Berlin, Germany, October 31-November 5, 1992
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.
Author: Carol Sue Dammel
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 200
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Book Description
Author: Cheryl L. Thomas
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 394
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Author: Chuck Merryman
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 332
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Author: Mirza A. Almehdi
Publisher:
ISBN:
Category : Ribosomes
Languages : en
Pages : 320
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Book Description
Ribosomes are multicomponent macromolecular particles and are essential for the survival of cells in all organisms. The ribosome's universal function is to catalyze polypeptide synthesis through translation of mRNA transcripts. Ribosomes from Escherichia coli, eubacterial organisms, have a sedimentation coefficient of 70S and are composed of 30S and 50S ribonucleoprotein subunits. The small ribosomal subunit is an assembly of 21 different proteins and a 16S ribosomal RNA. Within the 16S rRNA there are a few short stretches of universally conserved sequences spanning positions 517-533, 1394-1408, and 1492-1506. Clear functions for these sequence zones have not yet been assigned. Here I report a kinetic analysis of these highly conserved regions in the 16S rRNA and within the 30S ribosomal subunits. Binding affinity was measured in experiments that were based on protection from nuclease 51 digestion of short oligodeoxynucleotides hybridized to the designated regions. DNAs hybridized to regions 1400 and 1500 show significant differences in the apparent dissociation constants when measured in 30S particles as opposed to those found for 16S rRNA. Region 525 showed no difference in kinetic behavior. To further elucidate the functional and structural role played by the region centered about C1400 in 16S rRNA, a four nucleotide deletion was constructed within this region. The deletion was introduced by direct RNA manipulation using DNA/RNA hybridization, RNase H digestions, and ligation of the correct RNA fragments with T4 RNA ligase. I improved ligation efficiency of large RNA molecules by including a connector looped short DNA oligomer. Recycling products through phenyl boronate agarose (PBA-30) column also improved the efficiency of ligation. The mutagenized 16S rRNA fully reassembles into 30 particles and the altered 30S subunit possesses all of the normal ribosomal proteins. Altered ribosomes were functional in in vitro translation of MS2 mRNA. The altered ribosomes have lower translational activity relative to controls. Here I present indirect evidence suggesting that the decrease in the synthesis of MS2 coat proteins is the result of premature termination. The altered 16S RNA in ribosomes had an apparent dissociation constants with DNA probes comparable to those found for normal 16S rRNA. This suggest that the RNA is less flexible in the particle relative to normal 30S subunits. The deletion at 1400 did not have any effect on the physical properties of the 1500 region, as measured by DNA hybridization. A minor, but significant, effect on the 525 region was observed. A possible RNA/RNA interaction within the 30S particle is proposed to account for this observation.
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.
