By: Abdul-Rahman,Aamir, Sadam & Muhammad-Bux (BS III & Previous)

MOLECULAR BIOLOGYRNA polymerase functions and its modulation of activity.Termination , Gene expression and its regulation , Post translational Modifications.

RNA polymerase functions and its modulation of activity.

RNA polymerase functions: RNA polymerase (RNAP or RNApol),

also known as DNA-dependent RNA polymerase, is an enzyme that produces primary transcript RNA. In cells, RNAP is necessary for constructing RNA chains using DNA genes as templates, a process called transcription.

RNA polymerase is a huge factory with many moving parts. It is composed of a dozen different proteins. Together, they form a machine that surrounds DNA strands, unwinds them, and builds an RNA strand based on the information held inside the DNA. Once the enzyme gets started, RNA polymerase marches confidently along the DNA copying RNA strands thousands of nucleotides long.

RNA polymerase needs to be accurate in its copying of genetic information. To improve its accuracy, it performs a simple proof reading step as it builds an RNA strand. The active site is designed to be able to remove nucleotides as well as add them to the growing strand. The enzymetends to over around mismatched nucleotides longer than properly addedones, giving the enzyme time to remove them.

This process is some what wasteful, since proper nucleotides are also occasionally removed, but this is a small price to pay for creating better RNA transcripts. Overall, RNA polymerase makes an error about once in 10,000 nucleotides added, or about once per RNA strand created.

RNA polymerase: The enzyme that controls transcription and is characterized by:1. Search DNA for initiation site.2. It unwinds a short stretch of double helical DNA to produce a single-stranded DNA template.3. It selects the correct ribonucleotide and catalyzes the formation of a phosphodiester bond,4. It detects termination signals where transcript ends.

Modulation of RNA polymerase activity:Folding of the trigger loop of RNApolymerase promotes nucleotideaddition through creating a closed, catalytically competent conformation

of the active center. Here, we discuss the impact of adjacent RNA polymerase elements, including the F loop and the jaw domain, as well as external regulatory factors on the trigger loop folding and catalysis.Termination: After translation has been initiated the peptide chain is extended by the addition of further amino acid residues (elongation) until the

ribosome reaches a stop codon on the mRNA and the process is interrupted (termination).

When one of the three stop codons (UAA,UAG, or UGA) appears at the A site, termination starts.There are no complementary tRNAs forthe stop codons. Instead, two releasing factorsbind to the ribosome. One of these factors(RF–1) catalyzes hydrolytic cleavage of theester bond between the tRNA and the C–terminus of the peptide chain, thereby

Releasing the protein. Hydrolysis of GTP by factor RF–3 suppliesthe energy for the dissociation of the whole complex into its component parts. Energy requirements in protein synthesis are high. Four energy-rich phosphoric acid anhydride bonds are hydrolyzed for each amino acid residue. Amino acid activation uses up two energy-rich bonds per amino acid (ATP AMP + PP, and two GTPs are consumed per elongation cycle. In addition, initiation and

Stop Codons Are:UAA , UGA , UAG

termination each require one GTP per chain.Gene Expression: The process by which a gene's information is converted into the structures and functions of a cell by a process of producing a biologically functional molecule of either protein or RNA (gene product) is made.Gene expression is assumed to be controlled at various points in the sequence leading to protein synthesis. Gene Expression is done in two phases:

Transcription : Synthesis of an RNA that is complementary to one of the strands of DNA.Translation :Ribosomes read a messenger RNA and make protein according to its instruction.

Transcription

Translation

Regulation of gene expression :It includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA), and is informally termed gene regulation. Sophisticated programs of gene expression are widely observed in biology, for example to trigger developmental pathways, respond to environmental stimuli, or adapt to new food sources. Virtually any step of gene expression can be modulated, from -

transcriptional initiation, to RNA processing, and to the post-translational modification of a protein. Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed. Although as early as 1951, Barbara McClintock showed interaction between two genetic loci, Activator (Ac) and Dissociator (Ds), in the color formation of maize seeds, the first

discovery of a gene regulation system is widely considered to be the identification in 1961 of the lac operon, discovered by Jacques Monod, in which some enzymes involved in lactose metabolism are expressed by E. coli only in the presence of lactose and absence of glucose. Furthermore, in multicellular organisms, gene regulation drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types that possess different gene

expression profiles, and hence produce different proteins/have different ultrastructures that suit them to their functions (though they all possess the genotype, which follows the same genome sequence).Post translational modifications (PTM):It refers to the covalent and generally enzymatic modification of proteins during or after protein biosynthesis. Proteins are synthesized by ribosomes translating mRNA

into polypeptide chains, which may then undergo PTM to form the mature protein product. PTMs are important components in cell signaling.Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. They can extend the chemical repertoire of the 20 standard amino acids by introducing new functional groups such as phosphate, acetate, amide groups, or methyl groups.

Phosphorylation is a very common mechanism for regulating the activity of enzymes and is the most common post-translational modification. Many eukaryotic proteins also have carbohydrate molecules attached to them in a process called glycosylation, which can promote protein foldings and improve stability as well as serving regulatory functions. Attachment of lipid molecules, known as lipidation, often targets a protein or part of a protein to the cell membrane.

Other forms of post-translational modification consist of cleaving peptide bonds, as in processing a propeptide to a mature form or removing the initiator methionine residue. The formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two

polypeptide chains connected by disulfide bonds. Some types of post-translational modification are consequences of oxidative stress. Carbonylation is one example that targets the modified protein for degradation and can result in the formation of protein aggregates. Specific amino acid modifications can be used as biomarkers indicating oxidative damage. Sites that often undergo post-translational modifications are those that have a functional group that can serve as a

nucleophile in the reaction: the hydroxyl groups of serine, threonine, and tyrosine; the amine forms of lysine, arginine, and histidine; the thiolate anion of cysteine; the carboxylates of aspartate and glutamate; and the N- and C-termini. In addition, although the amides of aspargine and glutamine are weak nucleophiles, both can serve as attachment points for glycans. Rarer modifications can occur at oxidized methionines and at some methylenes in side chains.

Post-translational modification of proteins can be experimentally detected by a variety of techniques, including mass spectrometry, Eastern blotting, and Western blotting.

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