The Structural Heterogeneity of the 30S Ribosomal Subunit from Escherichia Coli PDF Download
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Author: Paul Voynow
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 190
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Book Description
Author: Paul Voynow
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 190
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Book Description
Author: Leonard Charles Lutter
Publisher:
ISBN:
Category :
Languages : en
Pages : 344
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Book Description
Author: Yu-Ju Meng
Publisher:
ISBN:
Category : Ribosomes
Languages : en
Pages : 232
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Book Description
Author: Eleanor Spicer Ward
Publisher:
ISBN:
Category : Ribosomes
Languages : en
Pages : 468
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Book Description
Author: Simon John Stollard Hardy
Publisher:
ISBN:
Category :
Languages : en
Pages : 374
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Author: Richard Louis Weiss
Publisher:
ISBN:
Category : Cations
Languages : en
Pages : 264
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Author: Seth Richard Stern
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 420
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Author: Jennifer Anne Maki
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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: Richard C. Conrad
Publisher:
ISBN:
Category :
Languages : en
Pages : 464
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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.
