We examined the translational activity of EF-Tu by using the PURE system, in vitro translation system derived from E.coli that was contains all the components essential for translation (17). Purified of wild-type EF-Tu from Synechocystis that had been treated with various concentrations of H2O2 were added to the translation system, which constructed without EF-Tu, and observed the synthesis of DHFR. The results revealed that the synthesis of DHFR was significantly decreased upon the increasing of H2O2 concentration (Fig. 1A). Only 0.1 mM H2O2 treated EF-Tu can inactivate the translational activity by decreased approximately 80% when compared with reduced EF-Tu (Fig. 1B). The results indicated that the translational activity of EF-Tu is hyper-sensitive to H2O2. By contrast, when we monitored translation in vitro of the mutated EF-Tu, which has the replacement of Cys82 by serine (isosteric to cysteine), on the sensitivity to oxidation by H2O2 showed the synthesis of DHFR was unaffected in the presence of H2O2 (Fig. 1C). Even though we added the concentration of H2O2 to 2 mM into the Cys82 mutant protein, the translational activity was quite constant, whereas the translational activity of wild-type EF-Tu was inactivation (Fig. 1B and 1D). These results suggested that the substitution of Cys82 by serine rendered EF-Tu insensitive to inactivation by H2O2 in translational machinery.
We monitored the redox state of Cys82 of EF-Tu by thiol-modification assay that modifying free thiol group with maleimidyl reagent (average molecular mass, 5 kDa). Modification of thiol group of reduced EF-Tu with this reagent resulted in the change of electrophoretic mobility of EF-Tu on a non-reducing SDS-PAGE by the molecular mass was increase when co...
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...wn as the G domain. It is involved in the formation of several hydrogen bonds with water molecules that coordinates the Mg2+ ion in the nucleotide binding pocket (22). Moreover, Cys81 is presence in the vicinity of the site of EF-Tu interaction with the 3′ end and 5′ end of the aminoacyl-tRNA molecule (23,24). The supporting data previously revealed that chemical modification of Cys82 by N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) in Thermus thermophilus EF-Tu (25,26), equivalent to Cys 81 of EF-Tu from E.coli (27,28) and Bacillus stearothermophilus (29), interfere with the formation of an appropriate pocket on EF-Tu to accommodate the aa-tRNA for ternary complex. Substitution of Cys82 by alanine prevents alkylation of EF-Tu by TosPheCH2Cl and allowed the interaction with aa-tRNA, suggesting that Cys82 plays a more specific role in the former conformation.
Miller, Kenneth R. and Joseph S. Levine. “Chapter 12: DNA and RNA.” Biology. Upper Saddle River: Pearson Education, Inc., 2002. Print.
The group of scientists hoped to determine the structure of the channel-forming domains in CFTR. The key experiment, called substituted-cysteine-accessibility method or S.C.A.M, consisted of mutating and substituting 9 consecutive residues in the M1 membrane spanning segment with cysteine in Xenopus oocytes, or eggs. If the mutated channels with cysteine still function, then they assumed that the structures of the mutated and normal channels were similar. Next, they determined the accessibility of the cysteine residue by adding the reagents MTSEA and MTSES, which are highly specific reagents that form a mixed disulfide with a free sulfhydryl covalently linking the reagent to the cysteine. In other words, if the MTSEA and MTSES bond with the cysteine residue and alter the conduction, they can assume the accessibility of the residue and then infer that the side chain of the corresponding wild type residue, or the residue before substitution, lines the channel. This process had been used to determining the structures of ion ch...
In molecular cell biology, restriction enzyme is used in various ways to understand the structure of a DNA molecule and how each of fragment of the sequence works. For this experiment, many researchers use several techniques and come up with logic questions that have various results to them. Such as in this study, researchers are analyzing the electroporation of BamHI or EcoRI together with pyr5-6 plasmids cuts with the same enzyme, which stimulates the efficiency of transformation in Dictyostelium discoideum (Kuspa and Loomis, 1992). This content shows the comparison between two different enzymes and how each fragments is affected with the source of pyr5-6 plasmid. Other studies use the three enzymes BamHI, EcoRI and HamIII together in numerous ways to get valuable results. Another technique is by constructing a restriction map, which is used in the lab.
J.C. Biro, B. Benyó, C. Sansom, Á. Szlávecz, G. Fördös, T. Micsik, and Z. Benyó; A common periodic table of codons and amino acids. Biochemical and Biophysical Research Communications 306 (2003) 408–415.
After the initiation process is complete, amino acids begin to be added to the polypeptide in a three step process known as elongation. First, the mRNA codon in the A site pairs with the anticodon of an incoming tRNA molecule. Next, the polypeptide separates from the tRNA in the P site and attaches to the amino acid that was carried by the tRNA in the A site. The ribosome catalyzes formation of the bond. Finally, the P site tRNA leaves the ribosome and the ribosome moves the tRNA in the A site to the P site with its attached polypeptide. A new tRNA is then able to bind to the A site to start the elongation process over again. Eventually, a stop codon will reach the A site signaling the amino acid to stop translation
(Kesssel,A. and Ben-Tal N. (2011) Introduction to Proteins: Structure, Function and Motion, London: CRC Press)
46- Kozak M. Rethinking some mechanisms invoked to explain translational regulation in eukaryotes. Gene. 2006; 382: 1-11.
The second phase involves the binding of the GroES co-chaperonin along with seven molecules of ATP in the presence of Mg2+ to a GroEL ring. Binding of GroES is dependent on the binding of ATP to the nucleotide-binding sites in the cis ring of GroEL. When these sites are fully saturated, the ATP molecules are free to enter and exit them without any steric obstruction. Weak binding of ATP to GroEL is sufficient to trigger a swift conformational change that allows rapid associated of GroES. The binding of GroES causes the residues of helices F and M of the intermediate domain to clamp onto the equatorial domain and close the binding sites in the cis ring.
Each of the nucleotides accommodate a phosphate group, sugar group, and a nitrogen base. There is four nitrogen bases in DNA. The four nitrogen bases are; Adenine (A), Thymine (T), Guanine (G), and Cytosine (C). Each of the bases are connected to a sugar molecule and a phosphate molecule. They are then positioned into two long strands that form a spiral called a double helix (DNA). The nitrogen bases are paired up with one another. Adenine and Thymine will always be paired with each other because of the bonds between them. Between A and T, there are two hydrogen bonds. The same goes with Guanine always being paired with Cytosine due. Between both G and C there is three hydrogen bonds. The nitrogen bases Adenine and Guanine won’t pair up with each other because, of their size. Both the nitrogen bases Adenine and Guanine are a purine base. Thymine and Cytosine are both a pyrimidine base. Adenine pairs with Thymine, and Guanine pairs with Cytosine, because they are of opposite
NMR experiments can provide useful structural information and probe different aspects of the molecule’s nuclear environment. Suggest two NMR experiments that may illustrate how a globular protein could respond to the changes of pH, temperature, and
The process of translation is a major part of protein synthesis. There are many different components related the process of protein synthesis which include the large ribosomal unit, 60S and the small ribosomal unit,40S. As well as these are the messenger RNA, “mRNA coding”, transfer RNA , tRNA for amino acids and finally greater than 12 of the catalytic proteins which have be found to be eIFs (eukaryotic initiation factors). (Norton and Layman, 2006)These initiation factors are quite important in relation to Protein Synthesis and translation initiation. ...“These initiation factors guide the assembly of the ribosome on the mRNA and are responsive to short-term changes in the availability of energy, amino acids, and growth factors. Initiation factors provide the cell with sensitivity to environmental factors, including changes in diet, such as leucine availability, and physical activity.”... As well as this they also enable the cell to become sensitive to factors like the availability of
Endonuclease V also known as EndoV is a highly conserved DNA repair enzyme. According to the research done in past, this enzyme was first discovered in E-coli and then later homologs of this enzyme were also found in prokaryotes, humans, and eukaryotes. This biochemical analysis of this protein is still not well understood by the scientist; however, Thermotoga maritime (Tma) which is a thermophillic bacterium is used as a great model to explain its enzymatic properties and structural properties.
The ETC carries out catabolic reactions that occur in the inner mitochondrial membrane. In the ETC hydrogen’s are removed during oxidation and are combined with the O2 to form water. The energy that is released from this reaction is utilized to attach phosphate groups to ADP, which forms the desired product of ATP. This process is defined as oxidative phosphorylation. Cofactors along the membrane of the mitochondria are the primary tools used for the ETC; these can be referred to as different complexes I-V. The reduced coenzymes NADH and FADH2 deliver the electrons to the first and second complex. These electrons are then transferred along the membrane from complex to complex each of which is reduced then oxidized. This pumps H+ into the intermembrane space, which creates an electrochemical gradient. The coenzyme ubiquinone helps to shuttle electrons between the larger complexes. At complex IV the electron pairs combine with protons and the formation of water occurs. Lastly at the ATP synthase complex the energy of the gradient is utilized to synthesize ATP. As the H+ flows between the membrane through the ATP synthase the rotator spins causing the phosphate to attach to ADP causing the formation of
Translation is when the information stored in the mRNA molecule is used to help create protein molecules. But, before this can take place, the mRNA must be read by ribosomes.
EDTA Titrations [homepage on the internet]. No date. [cited 2014 Mar 24]. Available from: http://bionmr.unl.edu/courses/chem221/lectures/chapter-12.ppt.