Native proteins generally function in a fully folded tertiary structure conformation in biological cells. In contrast, some native proteins have regions which are not properly structured also called as low-complexity domains (LC). For instance, RNA-binding FET family proteins, which include: Fused in sarcoma (FUS), Ewing’s sarcoma (EWS), and TATA-binding protein-associated factor (TAF15) have regions containing low complexity domains characterized by the abundance of only four amino acids; G, S, Q, &Y. FET family proteins are involved in many biological functions such as regulation of transcription, splicing, and mRNA export. Aberrant chromosomal translocations causes LCs of these FET family proteins fused to DNA-binding domains (DBD) of several other proteins which results in over expression of genes causing some types of cancers. Previous studies showed that LCs of FET family proteins activates transcription of genes when fused with DBD, but the mechanism of action of LCs in activating transcription is not known. McKnight et al. tried to unravel the mechanism of action of LCs in activating transcription. They showed that in order to activate transcription, LCs of FET family proteins should polymerize which helps them to bind to C-terminal domain (CTD) of RNA-polymerase II.
The authors obtained forty-three Y to S mutants of FUS LC domain (contains 27 repeats of triplet sequence [G/S] Y [G/S]) and fused them with GAL4 DBD. Through luciferase assays, they showed that fused proteins carrying one Y to S mutation activate transcription similar to that of wild type (wt) fusion protein, and as the number of Y to S mutations increases, transcriptional activation capacity of these mutants decreases, with one exceptional mutant called ‘2...
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... This observation gives evidence to the idea that polymerization of LC domains should precede their binding to CTD of RNA polymerase II.
In summary this study gave insight in to the mechanism of action of LC domains of FET proteins and RNA polymerase II. Experimental results in this study convincingly showed that polymerization of LC domains of FET proteins prompts their binding to CTD of RNA polymerase II; later phosphorylation of CTD causes its release from LCs, and this might be needed for transcriptional elongation by RNA polymerase II. This study is significant in our further understanding of the gene expression mechanisms. In future it is worth to see if the same mechanism of action is followed by other LC domain containing proteins, also it will be beneficial to look for some anti-cancer agents which can block polymerization of defective FET fusion proteins.
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.
Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping.
Ligation one was a 1:1 molar ratio pET-41a (+) vector: egfp insert that used 50ng NotI/NcoI cut pET-41a (+) DNA, 7ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Ligation two was a 1:3 molar ratio pET-41a (+) vector: egfp insert made up of 50ng NcoI/NotI cut pET-41a (+), 21ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Water was sterilized and deionized. The remaining three ligation samples served as controls. Ligation three contained 57ng uncut pET-41a (+)/EGFP recombinant plasmid DNA and sterile water. Ligation 4 was a negative control that consisted of only sterile water. Ligation five lacks DNA ligase but has the same properties of the 1:3 molar ratio pET-41a (+)/EGFP vector.
The expression of lac operon in each tube equals the amount of beta-galactosidase produced. Therefore, by looking at the amount of beta-galactosidase under different conditions collectively is a good way to understand the function of inducers and repressors in supervising the expression of lac operon and the control of gene expression generally.
In order to do this a polymer of DNA “unzips” into its two strands, a coding strand (left strand) and a template strand (right strand). Nucleotides of a molecule known as mRNA (messenger RNA) then temporarily bonds to the template strand and join together in the same way as nucleotides of DNA. Messenger RNA has a similar structure to that of DNA only it is single stranded. Like DNA, mRNA is made up of nucleotides again consisting of a phosphate, a sugar, and an organic nitrogenous base. However, unlike in DNA, the sugar in a nucleotide of mRNA is different (Ribose) and the nitrogenous base Thymine is replaced by a new base found in RNA known as Uracil (U)3b and like Thymine can only bond to its complimentary base Adenine. As a result of how it bonds to the DNA’s template strand, the mRNA strand formed is almost identical to the coding strand of DNA apart from these
The underlying purpose of the experiments performed in the study, Promoter Hypermethylation of KLF4 Inactivates its Tumor Suppressor Function in Cervical Carcinogenesis, is to investigate the mechanism by which the KLF4 gene is silenced in cervical carcinomas. Cervical cancer accounts for 250,000 female deaths every year. Developing therapies for cervical cancer has been limited due to the lack of genetic and epigenetic data of the mechanism causing the cancer. The KLF4 gene is a transcriptional regulator of cell growth and differentiation. It functions as a tumor suppressor in cervical cancer, but is found to be inactivated in cervical cancer. The overexpression of KLF4 protein is known to inhibit cervical cancer cell growth and tumor formation by activating a cell cycle suppressor. Promoter CpG island hypermethylation can result in transcriptional silencing of many tumor suppressing genes. Two CpG regions, BSQ1 and BSQ3, were examined in this experiment.
In the field of genetics, the study of the effect of various genes is imperative in translation and interpretation. As genetic coding influences phenotypic expression, the analysis of specific genes and any polymorphisms are relevant in a clinical setting. One such example is that of personality traits, which are believed to be influenced by specific neurotransmitters, known as catecholamines. Catecholamines are chemicals released by the adrenal glands in response to stress, and operate dually as hormones and neurotransmitters within the body. Commonly, catecholamines mediate functions within the central nervous system, including those of emotional responses and motor control. Inclusive of dopamine, epinephrine and norepinephrine, the secretion and metabolism of these chemicals is thought to impact upon various mental functions and behaviours.
RNAi involves complementary base-pairing with the target RNA to bring about repression, while DNA and chromatin modification requires bromodomains, chromodomains, specific amino-acid residues and chemical groups for protein- DNA and -histone interaction.
Gene expression is the ability of a gene to produce a biologically active protein. This process is regulated by the cells of an organism, it is very important to the survival of organisms at all levels. This is much more complex in eukaryotes than in prokaryotes. A major difference is the presence in eukaryotes of a nuclear membrane, which prevents the simultaneous transcription and translation that occurs in prokaryotes. Initiation of protein transcription is started by RNA polymerase. The activity of RNA polymerase is regulated by interaction with regulatory proteins; these proteins can act both positively, as activators, and negatively as repressors. An example of gene regulation in cells is the activity of the trp operon. The trp operon encodes the genes for the synthesis of tryptophan. This type of gene, like the lac operon, is regulated by a repressor that binds to the operator sequences. The activity of the trp repressor is enhanced when it binds tryptophan; in this capacity, tryptophan is known as a corepressor. Since the activity of the trp repressor is enhanced in the presence of tryptophan, the rate of expression of the trp operon is graded in response to the level of tryptophan in the cell. Another example of gene regulation in cells is gene amplification. This is a Technique by which selected DNA from a single cell can be duplicated indefinitely until there is a sufficient amount to analyse by conventional genetic techniques.
Proteins called transcription factors, however, play a particularly central role in regulating transcription. These important proteins help determine which genes are active in each cell of your body.
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
To form a polynucleotide DNA, many nucleotides are linked together with 3`-5` phosphodiester linkages. In a compl...
Almost all biology students learn the fundamentals of gene expression, DNA contains information which is transcribed into RNA to create protein. Students however, are not taught of RNA Interference, the biological process where RNA molecules inhibit a gene’s expression, RNAi for short. While RNAi is a fairly new discovery, its use in modern biological research is groundbreaking. RNA Interference works by binding Double-stranded RNA molecules (siRNA) to a complementary messenger RNA. The enzymes Dicer and Slicer then cleave the chemical bonds which hold the messeger RNA in place and prevent it from delivering protein silencing instructions thus, the term, Gene Silencing. This phenomenon was first discovered by Richard Jorgensen in 1990 when he was trying to produce deeper purple colored petunias by introducing more purple pigment genes to the flower. To his surprise, the purple petunia turned completely white and got the opposite of his predicted result. At the time Jorgensen coined this effect, “Cosuppression”. It was not until 1998 that Andrew Fire and Craig, C Mello explained the process of RNAi and discovered its use in Caenorhabditis elegans (C. Elegans). In 2006 Fire and Mello won the Nobel Prize in Physiology or Medicine “for their discover of RNA Interference – gene silencing by double stranded RNA”. They utilized the nematode, C. Elegans due to its whole genome being sequenced. This unique characteristic allows for every gene to be tested
Distinct characteristics are not only an end result of the DNA sequence but also of the cell’s internal system of expression orchestrated by different proteins and RNAs present at a given time. DNA encodes for many possible characteristics, but different types of RNA aided by specialized proteins sometimes with external signals express the needed genes. Control of gene expression is of vital importance for an eukaryote’s survival such as the ability of switching genes on/off in accordance with the changes in the environment (Campbell and Reece, 2008). Of a cell’s entire genome, only 15% will be expressed, and in multicellular organisms the genes active will vary according to their specialization. (Fletcher, Ivor & Winter, 2007).
Zinc is a cofactor in over 300 metalloproteins including RNA and DNA polymerase, thus indispensable part for protein synthesis, DNA synthesis, and cell growth. Zinc proteins are estimated to be around about 10% of the human proteome.[12] The essentiality of zinc in the formation of enzymes was first demonstrated in 1940 when zinc was found in the composition of the carbon anhydrase enzyme [13], ever since studies have been carried out on different classes of enzymes and proteins to understand the role of zinc in composition and functions of