The proteins that help modulate DNA functions are DNA binding proteins. Its functions include controlling protein production, regulating cell growth and division and storing DNA inside nucleus. The transcription process involves the specific site of DNA in which attaches a DNA binding protein are transcription factors, it is also called a promotor. The transcription factors help RNA polymerase to start DNA transcription. Most proteins that activate or inhibit RNA pol II have two functional domains- DNA binding domain and transcription activator domain. DNA binding domains have domain that are helix turn helix, zinc fingers, basic region leucine zipper. DNA binding proteins are specific binding of proteins to a variety of nucleic acids underlies all aspects of gene expression, including genome replication, repair, transcription and RNA metabolism. RNA POLYMERASE RNA polymerase is the ribonucleic acid polymerase or DNA directed RNA polymerase. The double stranded DNA is opened to separate two strands by RNA polymerase in which one strand of exposed nucleotides is used for as template for the synthesis of RNA, a process called transcription. In promoter region the transcription factor and the transcription mediator complex attach to DNA binding site before the initiation of DNA unwinding by RNA polymerase. It not only initiate transcription, also guides nucleotides in it position, facilitates attachment and elongation, has intrinsic proofreading and replacement capabilities and termination recognition capability.EUKARYOTIC RNA POLYMERASEEukaryotic RNA polymerase has three types I, II, III which are responsible for synthesis of particular classes of RNA.RNA polymerase I makes only r RNA, RNA polymerase II synthesizes all m RNA, and RNA pol III makes t RNA and 5s RNA of ribosomes. all are found in the nucleus. Each is higher molecular weight. The class II enzymes are highly sensitive to inhibition by alpha amanitin. The class I enzymes are resistant to alpha amanitin inhibitor. Each of the enzymes are isolated in a variety of chromatographically and electrophoretically distinct forms. E.coli RNA POLYMERASEE.coli RNA polymerase has five subunits – two identical ? subunits and one each of types ?, ?’ and ?. It is the biggest enzyme known with a total molecular weight of 465,000. E.coli mutants containing altered subunit structures have been isolated. The ? subunit dissociates from the enzyme during the elongation stage of RNA polymerization. The term core enzyme is used to describe the ? free unit namely ?2??’ the complete enzyme, ?2??’? is called the holoenzyme. SYNTHESIS OF RNAThe chemical characteristics of the synthesis of RNA :The precursor in the synthesis of RNA are the four ribo nucleoside 5′-triphosphate (NTP) ATP, GTP, CTP, UTP. On the ribose portion of each NTP there are two -OH group- one each on the 2′- and 3′-carbon atomIn the polymerization reaction a 3′ -OH group of one nucleoside reacts with the 5′ triphosphate of a second nucleoside. Pyrophosphate is removed and a phosphodiester bond results. This is the same reaction that occurs in the synthesis of DNA.The sequence of base in an RNA molecule is determined by the base sequence of the DNA. Each base added to the growing end of the RNA chain is chosen by its ability to base pair with the DNA strand used as a template; thus, the bases C, T, G and A in a DNA strand cause G, A, C and U, respectively, to appear in the newly synthesized RNA molecule.The DNA molecule being transcribed is double stranded and in a particular region only one strand serve as a template.The RNA chain grows in the 5′ to 3′ directions that is nucleotides are added only to the 3′ -OH end of the growing chain this direction of chain growth is the same as that in DNA synthesis RNA polymerases, in contrast with DNA polymerase are able to initiate chain growth, no primer is needed.Only ribo nucleoside 5′-Triphosphates participate in RNA synthesis and the first base to be laid down in the initiation event is a Triphosphate. Its 3′-OH group is the point of attachment of the subsequent nucleotide. Thus the 5′ end of growing RNA molecule terminate with Triphosphate. The overall polymerization reaction may be written as The synthesis of RNA consists of four discrete stages:1) Binding of RNA polymerase to a template at specific site. 2) Initiation 3) Chain elongation4) Chain termination and release.TATA BOXA TATA BOX is a conserved sequence found in the promoter region which is responsible for the binding of transcription factors or histones. It is usually located approximately 25 base pairs upstream of the coding region. It is example of a cis-regulatory element which has core sequence. The TATA BOX is found approximately 24% of human genes. The TATA region is normally bound to a TATA binding protein, a transcriptional factor that interacts with other transcription factors to form a pre initiation complex for the binding of RNA polymerase. The DNA unwinding process starts at this point.RHO FACTOR (?)The prokaryote protein involved in termination of transcription is RHO factor. It binds to transcription terminator pause site, an exposed region of single stranded RNA. there are two transcription termination in prokaryotes :- i) ?- dependent termination, ii) ?- independent termination. The ? factor also act as RNA substrate.SIGMA FACTOR (?)The sigma factor is a protein only for transcription initiation. It enables specific biding of RNA polymerase to promote genes. Selection of promoters by RNA polymerase is dependent in the sigma factor that dissociate with it. The RNA polymerase holoenzyme contains exactly one sigma factor subunit. Sigma factors are characterized by their molecular weight.PROCESS OF TRANSCRIPTIONThe transcription process involves three stages, i)Initiation, ii)Elongation and iii)Termination.The first step in transcription is the binding of RNA polymerase to a DNA molecule. Binding occurs at a particular region called a promoter which are sequence in which several interactions occur. Several events occur at a promoter; RNA polymerase must recognize a specific DNA sequence, attach in a proper confirmation, locally open the DNA strand in order to gain access to the bases to be copied, and then initiate synthesis. These events are guided by the base sequence of the DNA, the polymerase ? subunit and, for some promoters, also by auxiliary proteins.INITIATIONThe RNA polymerase is ready to initiate synthesis once the open promoter complex is formed. RNA polymerase has two nucleotide binding sites called the initiation site and the elongation site. Thymine is the first DNA base to be transcribed. The initiating nucleoside triphosphate binds to the enzyme in the open promoter complex and forms a hydrogen bond with the complementary DNA base. The RNA pol core enzyme binds to the another subunit called the sigma subunit to form a holoenzyme capable of unwinding the DNA double helix in order to facilitate access to gene. The promoter specificity to RNA polymerase was conveyed by sigma subunit where it has to bind. enhancer sequence acts at which gene to be transcribed. ELONGATIONOnce transcription is initiated the template strand is read by RNA polymerase. After several nucleotides are added to the growing chain, RNA polymerase undergoes conformational change and loses the ? subunit. This marks the transition from the shuttering of the initiation phase, to the stable forward movement of the elongation phase. Most elongation is carried out by the core enzyme. The core enzyme moves along the DNA, binding a nucleoside triphosphate that can pair with the next DNA base and opening the DNA helix as it moves. The DNA helix recloses as synthesis proceeds. The newly synthesized RNA is released from its hydrogen bonds with the DNA in the open region. The chain elongation does not occur at a constant rate, which means synthesis markedly slows down when particular regions of DNA are passed, then continues at a normal rate, slows down, accelerates again and so forth. This reduction in rate is called pause. Analysis of pausing along stretches of DNA of known sequence shows that pausing frequently follows sequence that forms hairpins in the RNA, but at least half of the pause sites have no recognizable features. TERMINATIONTermination of RNA synthesis occur at specific base sequences within the DNA molecule. These sequences are of two types, simple terminators and those that require auxiliary termination factors. At non-coding sequence the termination sequence is found. Termination occurs by rho-dependent and rho-independent termination. In rho- independent termination, inverted repeats sequences are transcribed in which the sequence fold themselves to hairpin loop ad pause the RNA polymerase thus release the transcript.In rho-dependent termination it uses rho factor which unwinds DNA-RNA hybrid actively at transcription and therefore releases the newly synthesized RNA.REGULATIONTranscription regulation is the process by which a cell controls the expression of information encoded in the DNA, and is able to excise control over gene activity. It is like turning on or turning off the production of proteins, by regulating transcription of the relevant RNA. this allows the cell to respond appropriately to various signals. Transcriptional regulation depends both on the accessibility of DNA that needs to be transcribed, as well as the action of proteins called transcriptional factors.The default state of transcription in higher organisms including human being is off, which means that unless transcription is turned on, gene expression cannot occur. this packing and unpacking of DNA is another level at which transcription can be controlled. Transcription and translation are events that take place in two different locations in the cell. While transcription occurs in the nucleus, translation cannot occur in cytoplasm, unless the transcribed m RNA is able to leave the nucleus and enter the cytoplasm.TRANSCRIPTION IN PROKARYOTES AND EUKARYOTESRNA is synthesized by RNA polymerase. The primary transcripts thus produced must usually be processed further before they can be used by the cell. prokaryote m RNAs are short lived and are often translated into protein at one end while the other end is still transcribed. The length of these m RNAs is heterogenous; some m RNAs code for several proteins. All prokaryotic RNAs are transcribed by the same enzyme, which is a large and complex multi-subunit molecule consisting of several polypeptides. One of the peptides, the sigma factor, is loosely bound to the core polymerase and is required for the enzyme to recognize the correct start signals on the DNA. Eukaryotic transcription takes place in nucleus; eukaryote m RNAs are considerably more stable than those of prokaryotes are monocistronic. Three nuclear RNA polymerases have been identified. Each of which transcribes a different classes of genes. These polymerases an be distinguished by their order of elution from ion-exchange columns and by their sensitivity to alpha amanitin. RNA SPLICINGA characteristic of most of the primary transcripts of higher eukaryotes and some transcripts in all eukaryotes is the presence of untranslated intervening sequence that interrupt the coding sequence and are excised from the primary RNA transcript. In the processing of RNA in higher eukaryotes the amount of discarded RNA ranges from 50 to nearly 90 percent of the primary transcript. the remaining segments are joined together to form the finished m RNA molecules. The excision of introns and the formation of the final m RNA molecule by joining of the exons is called RNA splicing. Splicing is accomplished with the help of spliceosomes, which remove introns from the genes in RNA. Spliceosomes are composed of a mixture of protein and small RNA molecules. They locate the ends of the introns, cut them away from the exons, and joins the ends of adjacent exons together. Once the entire gene is devoid of its introns, the process of RNA splicing is complete. Alternative splicing is the single gene coding for more that one protein. The time for m RNA to move out of the nucleus is when all the introns are spliced from gene from which the translation begins. RNA interferenceRNA interference is also called RNAi or post transcriptional gene silencing. After transcription it is the process results down- regulation of a gene at the RNA level. There is also gene silencing at the transcriptional level(TGS). Examples :- transposons, retroviral genes, heterochromatin.RNA silencing is a novel gene regulatory mechanism that limits the transcript level by either suppressing transcription or by activating a sequence-specific RNA degradation process. After they are produced by transcription, the small interfering RNA molecules fold into double-stranded hairpin loops.An enzyme called ‘DICER’ enzyme cuts, or dices, these double-stranded loops into microRNA, each about 20 base pairs in length. The two strands of the loops then separate.CONCLUSIONTranscription begins with initiation, which requires a promoter, a special sequence of DNA to which the RNA polymerase binds very tightly. A promoter orients the RNA polymerase and thus aims at the correct strand to use as a template. Part of each promoter is the initiation site, where transcription begins. Once RNA polymerase has bound to the promoter, it begins the process of elongation. RNA polymerase unwinds the DNA about 10 base pairs at a time and reads the template strand in the 3′ to 5′ direction while it forms RNA in the opposite direction. As in the process of DNA replication, base pairing rules apply. However, uracil is used in RNA instead of the base thymine, which is used in DNA. The base sequence specify its termination.
All Research Proposal
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