Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

scholarly journals Biophysical characterization of the structure and flexibility of the E. Coli ribosome

2019 ◽  
Author(s):  
◽  
Emily Doris Armbruster
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
Structure And Function ◽  
23S Rrna ◽  
Internal Cavity ◽  
Macromolecular Complex ◽  
Biophysical Characterization ◽  
E Coli ◽  
Bacterial Ribosome ◽  
And Function

At the discovery of ribosomes by George Palade in 1955 in the first image of the subcellular environment, he described them as "a particulate component of small dimensions (100 to 150 [alpha]) and high density". Subsequently, the ribosome was shown to be the site of protein synthesis, or translation, and thus an essential macromolecular complex for all cells. Ribosomes can have variability from species to species, but the overall structure and function are conserved [66]. Ribosomes are named according to their sedimentation coefficients, a unit of density expressed in Svedbergs (abbreviated S). The three most studied and most prevalent ribosomes are the bacterial 70S, the eukaryotic 80S, and the 55S mitoribosome, which is present in the mitochondrion organelle. The bacterial ribosome serves as a target for many antibiotics and is a model system for investigating the structure and function of this "nanomachine". Despite variations in size, all ribosomes consist of a small and a large subunit that when bound together have an internal cavity that is divided into three sites, named A, P, or E site. The bacterial ribosome has a 30S small subunit, which consists of a 16S rRNA and 21 attached proteins, and a 50S large subunit that is made up of the 23S rRNA, 5S rRNA and 31 proteins. This dissertation discusses the 70S bacterial ribosome, other than when the 80S eukaryotic ribosome is specified.

scholarly journals Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (ββ′) of Saccharomyces cerevisiae Ribonucleotide Reductase

2013 ◽  
Vol 288 (20) ◽  
pp. 13951-13959 ◽  
Author(s):  
Yan Zhang ◽  
Xiuxiang An ◽  
JoAnne Stubbe ◽  
Mingxia Huang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Small Subunit ◽  
Cluster Assembly ◽  
E Coli ◽  
Viability Assay ◽  
Docking Model

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.

scholarly journals Conserved Structure and Function in the Granulysin and NK-Lysin Peptide Family

2005 ◽  
Vol 73 (10) ◽  
pp. 6332-6339 ◽  
Author(s):  
Charlotte M. A. Linde ◽  
Susanna Grundström ◽  
Erik Nordling ◽  
Essam Refai ◽  
Patrick J. Brennan ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Three Dimensional ◽  
Antimycobacterial Activity ◽  
Structure And Function ◽  
Loop Structure ◽  
E Coli ◽  
Short Loop ◽  
Peptide Family ◽  
And Function ◽  
Related Proteins

ABSTRACT Granulysin and NK-lysin are homologous bactericidal proteins with a moderate residue identity (35%), both of which have antimycobacterial activity. Short loop peptides derived from the antimycobacterial domains of granulysin, NK-lysin, and a putative chicken NK-lysin were examined and shown to have comparable antimycobacterial but variable Escherichia coli activities. The known structure of the NK-lysin loop peptide was used to predict the structure of the equivalent peptides of granulysin and chicken NK-lysin by homology modeling. The last two adopted a secondary structure almost identical to that of NK-lysin. All three peptides form very similar three-dimensional (3-D) architectures in which the important basic residues assume the same positions in space. The basic residues in granulysin are arginine, while those in NK-lysin and chicken NK-lysin are a mixture of arginine and lysine. We altered the ratio of arginine to lysine in the granulysin fragment to examine the importance of basic residues for antimycobacterial activity. The alteration of the amino acids reduced the activity against E. coli to a larger extent than that against Mycobacterium smegmatis. In granulysin, the arginines in the loop structure are not crucial for antimycobacterial activity but are important for cytotoxicity. We suggest that the antibacterial domains of the related proteins granulysin, NK-lysin, and chicken NK-lysin have conserved their 3-D structure and their function against mycobacteria.

scholarly journals The structure and function of tRNA-like domain in E. coli tmRNA

2000 ◽  
Vol 44 (1) ◽  
pp. 263-264
Author(s):  
K. Hanawa ◽  
S. Lee ◽  
H. Himeno ◽  
A. Muto
Keyword(s):  
Structure And Function ◽  
E Coli ◽  
And Function

Comparison of the structure and function of ribulosebisphosphate carboxylase–oxygenase from a cold-hardy and nonhardy potato species

1981 ◽  
Vol 59 (4) ◽  
pp. 280-289 ◽  
Author(s):  
Norman P. A. Huner ◽  
Jiwan P. Palta ◽  
Paul H. Li ◽  
John V. Carter
Keyword(s):  
Large Subunit ◽  
Structure And Function ◽  
Sulfhydryl Groups ◽  
Relative Mass ◽  
Nitrobenzoic Acid ◽  
Significant Difference ◽  
Ribulosebisphosphate Carboxylase ◽  
Cold Hardy ◽  
And Function ◽  
Kinetics Of

A comparison of ribulosebisphosphate carboxylase–oxygenase from the leaves of the non-acclimated, cold-hardy species, Solanum commersonii, and the nonacclimated, nonhardy species, Solanum tuberosum showed that this enzyme from the two species differed in structure and function. The results of sulfhydryl group titration with 5,5′-dithiobis(2-nitrobenzoic acid) indicated that the kinetics of titration and the number of accessible sulfhydryl groups in the native enzymes were different. After 30 min, the enzyme from the hardy species had 1.7 times fewer sulfhydryl groups titrated than that from the nonhardy species. In the presence of 1% (w/v) sodium dodecyl sulfate, the total number of sulfhydryl groups titratable with 5,5′-dithiobis-(2-nitrobenzoic acid) was the same for both species. However, this denaturant had a differential effect on the kinetics of titration with 5,5′-dithiobis(2-nitrobenzoic acid). Both enzymes had a native molecular weight of about 550 000. The quaternary structures of the two enzymes were similar with the presence of large and small subunits of 54 000 and 14 000, respectively. However, there was more polypeptide of 108 000 – 110 000 present in preparations of the enzyme from S. tuberosum than from S. commersonii. This polypeptide is an apparent dimer of the large subunit on a relative mass basis. The large subunit of the enzyme from S. tuberosum was more sensitive to the absence of reducing agent and was more sensitive to freezing and thawing than the large subunit of the enzyme from S. commersonii. Catalytic properties of both enzymes at 5 and 25 °C indicated no significant difference in the [Formula: see text] at either temperature. However, the Vmax at 5 °C for the enzyme from S. commersonii was 35% higher than that of the enzyme from S. tuberosum. In contrast, the Vmax at 25 °C for the enzyme of the hardy species was 250% lower than that of the enzyme from the nonhardy species.

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