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.
Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
The goal of this two week lab was to examine the stereochemistry of the oxidation-reduction interconversion of 4-tert-butylcyclohexanol and 4-tert-butylcyclohexanone. The purpose of first week was to explore the oxidation of an alcohol to a ketone and see how the reduction of the ketone will affect the stereoselectivity. The purpose of first week is to oxidize the alcohol, 4-tert-butylcyclohexanol, to ketone just so that it can be reduced back into the alcohol to see how OH will react. The purpose of second week was to reduce 4-tert-butylcyclohexanol from first week and determine the effect of the product's diastereoselectivity by performing reduction procedures using sodium borohydride The chemicals for this lab are sodium hypochlorite, 4-tert-butylcyclohexanone
The two diastereomeric products that are made do not separate well by TLC. In order to see the two isomers, it is necessary to convert the products into derivatives. To more easily identify the stereoisomers produced in the reduction of benzil, hydrobenzoin is converted to acetonide by reacting with 2-methoxypropene and acid. The cis and trans isomers of these derivatives have different Rf values on TLC making it possible to identify the stereoisomers that have been prepared. The solution is compared with syn and anti acetonide on two different TLC plates.
Compounds resulting from heterocyclic chemistry form a huge family of variations of organic compounds with cyclic or acyclic structures having carbons and at least another element with the most common being sulfur, oxygen, fluorine or nitrogen 49. Such heterocyclic compounds must have their rings having heteroatoms and the rings could be unsaturated or saturated as well 1. These compounds are usually classified as aromatic or alicyclic heterocycles 6. The latter contain cyclic analogues of thioethers, ethers and amines 6. It has been studied before that the families of nitrogen- sulfur heterocycles have aromatic compounds that are very stable19. Also, fluorinated compound using trifluoromethyl group is often used to reduce in vivo metabolism of potential drugs, in addition to improving cellular membrane permeability (LogP)88. Sulfur and Nitrogen organic compounds heterocycles are basically drawn from aromatic heterocycles realized when a heteroatom occupies the position of a carbon atom in the ring of an organic aromatic
Transformation of T87 cells was done by culturing the cells in B5 medium supplemented with 1 μM 1-naphthaleneacetic acid (NAA) and 40 g L-1 sucrose. The cells were cultured for one day at 22°C with continuous illumination and shaking at 120g. Next, 10 μL of overnight cultured Agrobacterium transformed with respective vectors were added into the cell suspension and cultured for an additional two days. After co-cultivation, the cell suspension was washed thrice with 10 mL of JPL3 medium supplemented with Carbencilin (250 μg mL-1) by centrifuging at 100g for two minutes. Finally, the cells were resuspended and spread onto JPL3 selection agar plate supplemented with Carbencilin (250 μg mL-1), Kanamycin (30 μg mL-1) an...
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...
The operon is a set of coding regions of DNA clustered together that includes structural genes and it is under the control of a single regulatory region. The operator regulates transcription, which is a repressor protein. When the operator binds to a segment of the regulatory region, transcription is shut down.
EDTA, the chelating agent that binds with magnesium, had a high absorbency and strong color change to red. The correct cofactor was copper which with the chelating agent of PTU and citric acid which both bind strongly to copper which keeps it from binding with the enzyme. This was determined because in the trails, both PTU and citric acid had low absorbency and were clear or roughly clear in color. The catechol in each tube, which was the control for this experiment, allowed the cofactor that would be used in this reaction to be singled out. The way each chelating agent would affect the different cofactors displayed which was not needed for the reaction and which cofactors were needed for the reaction. An inconsistency that may have affected the data would be if the calibration tube malfunctioned in balancing the spectrophotometer to zero. There also could be errors if the calibration tube wasn’t used before each tube was tested in the spectrophotometer. The relationship of the cofactor and amount of enzyme activity would be that if the cofactor is inhibited or not, the enzyme activity would be higher if the cofactor is not inhibited but lower if it was inhibited by the chelating
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.
There are some significant differences between Archaea and the two other domains in terms of structure, which creates an advantageous heat resistance in Archaea. Bacteria and eukaryotes both have ester linkages between hydrophobic side chains and glycerol in the membrane, whereas archaea have ether bonds instead and lack true fatty acid side chains, instead having 40-carbon phytane chains. Additionally, the di...
There are three main divisions of living organisms: Prokaryotes, eukaryotes and archaea. This essay will outline the division between the prokaryotic and eukaryotic organisms and explore the reasoning behind such differences with regard to general structure, storage of deoxyribonucleic acid and its replication, metabolic processes, protein synthesis and ribonucleic acid processing.
Thus, it contains two active sites and has metal ions that play a key role in regulating catalytic activities and stabilizing enzyme-substrate complex. The active sites are 30Å apart from each other (7). As proposed by Gettins and Coleman using NMR studies (6), each active site of AP comprises of three metal binding sites, which acknowledged as M1, M2, and M3. Two zinc ions bind to M1 and M2 sites while a magnesium ion occupies M3. In each active site, two zinc ions are 4Å apart while magnesium ion is 5Å and 7Å away from two zinc ions (7). The structures of AP are shown in Figure 1, which specifically indicating metal binding sites and interaction with essential amino acids in active
A previous study, looked at by the researchers, stated that nuclear localization signals are what allow the RNA to enter the nucleus (Wu W, Pante N. 2009). This persuaded them to ask the question of whether or not there was a nuclear localization signal within a viral protein of HCRSV. The localization of P23 was then tested using a transient expression method. The results of their experiment showed that there was a strong signal detected in the nucleus of the Kenaf leaf samples. This proved that P23 was in fact localized in the nucleus and that a nuclear localization signal is present in P23 (Gao R, Liu P, Wong SM. 2012). It was also found that P23 has the ability to bind to carrier proteins that come into the nucleus. This showed that even if P23 was not localized in the nucleus, it could still enter. The mode of entry into the nucleus was discovered to by α-importin (Gao R, Liu P, Wong SM. 2012) . This was discovered by experimenting with a probe of anti-importin α antibody. α-importin was only detected in the protein extract of P23 in the nucleus of the HCRSV-infected Kenaf sample (Gao R, Liu P, Wong SM. 2012). Researchers concluded from their results of the experiments that α-importin, P23, and HCRSV RNA form a complex that enters the nucleus to begin replication of the
EDTA Titrations [homepage on the internet]. No date. [cited 2014 Mar 24]. Available from: http://bionmr.unl.edu/courses/chem221/lectures/chapter-12.ppt.