RNA SPLICING:

The protein coding genes of eukaryote typically contain regions of DNA that serves no coding function. Non coding regions called introns interrupt the coding regions called exons. When the gene is transcribed into RNA, both coding and non coding regions are copied. RNA splicing is a modification of RNA after transcription, in which introns are removed and exons are joined. Typical eukaryotic messenger RNA should be modified before it can be used to produce a correct protein through translation. RNA splicing occurs inside the nucleus before the RNA migrates into cytoplasm. The type of splicing depends on the structure of the spliced introns and the catalysts required for splicing to occur. Splicing is catalyzed by the spliceosome which is a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs, pronounced snurps), but there are also self-splicing introns.. The RNA components of snRNPs interact with the introns and may be involved in catalysis. Spliceosomal introns often reside in eukaryotic protein-coding genes. Within the introns, a 3' splice site, 5' splice site, and branch site are required for splicing. The 5' splice site or splice donor site includes an almost invariant sequence GU at the 5' end of the introns, within a larger, less highly conserved consensus region. The 3' splice site or splice acceptor site terminates the introns with an almost invariant AG sequence. snRNPs bind to sites in pre mRNA at or near the intron exon boundaries. The snRNPs contain RNA molecules that can bind to the sequence through complementary base pairing. The snRNPs bound to the consensus sequences and form a large complex called spliceosome. As the spliceosome forms, intron loops out. The spliceosome cuts the pre mRNA at one intron exon boundary, where it leaves a reactive free hydroxyl group at exon. The spliceosome uses this hydroxyl group to attack the other end of intron, and in this process introns are removed and exons are joined forming a mature RNA molecule. The mRNA molecule leaves the cell and is translated into protein within cytoplasm. The introns are degraded quickly and the snRNPs remain in the cell which is used to splice the introns from other RNA molecules. Self-splicing occurs for rare introns that form a ribozyme, performing the functions of the spliceosome by RNA alone. There are three kinds of self-splicing introns, Group I, Group II and Group III. Group I and II introns perform splicing similar to the spliceosome without requiring any protein.

TRANSCRIPTION FACTORS:
A transcription factor (sometimes called a sequence-specific DNA-binding factor) is a protein that binds to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to mRNA. Transcription factors perform this function alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes. A defining feature of transcription factors is that they contain one or more DNAbinding domains (DBDs), which attach to specific sequences of DNA adjacent to the genes that they regulate. Transcription factors bind to either enhancer or promoter regions of DNA adjacent to the genes that they regulate. Depending on the transcription factor, the transcription of the adjacent gene is either up- or down-regulated. Transcription factors use a variety of mechanisms for the regulation of gene expression. These mechanisms include:

stabilize or block the binding of RNA polymerase to DNA catalyze the acetylation or deacetylation of histone proteins. The transcription factor can either do this directly or recruit other proteins with this catalytic activity

General transcription factors are required for transcription in eukaryotes from all genes. GTFs assist RNA Pol II in transcription initiation and are designated TFIIA, TFIIB,... and most of them are multimeric proteins.Equivalent GTFs are highly conserved among the eukaryotes. In prokaryotes, only one general transcription factor, known as s factor is required. TFIID Composed of 14 subunits, one of them being TATA box binding protein, TBP Functions: promoter recognition, TFIIB recruitment TFIIB Start site selection for Pol II TFIIF-PolII complex recruitment Some TFIIB mutants result in a shift of transcription start site Under certain conditions (BRE element promoter) Pol II together with TFIID and TFIIB can form the minimal initiation complex. At most promoters, however, TFIIE, F and H are necessary TFIIF - Recruitment of Pol II to the existing DNA-TFIID-B complex, - Positioning Pol II over the start site - Binding to the non-template DNA strand. - TFIIF also reduces non-specific binding of RNA pol II to DNA.

TFIIE TFIIE is a heterotetrameric protein ( ) 2 - TFIIE appears to create the docking site for next transcription factor, TFIIH. - TFIIE also modulates TFIIH enzymatic activities - In addition TFIIE enhances promoter melting. TFIIH TFIIH is a multimeric protein, composed of 9 subunits, some of them with distinct enzymatic activities - TFIIH has a helicase activity, which unwinds the DNA duplex at a start site, allowing Pol II to bind to the template strand. - TFIIH also has a kinase ativity, it phosphorylates PolII in the begining of elongation Other TFIIH subunits have been shown to recruit DNA-repairing enzymes if polymerase reaches damaged region in DNA and gets stalled. TFIIA For transcription in vivo, another factor TFIIA is required The function of TFIIA is somewhat unclear, but it might help the other factors to TFIIA has also shown to have some anti-repressor functions TFIIA is not required for transcription in vitro.

bind.

RNA PolII cannot initiate transcription itself, but is absolutely dependent on auxiliary transcription factors (called TFIIX, where "X" is a letter that identifies the individual factor). The first step in the complex formation at a promoter containing a TATA box is binding of the factor TFIID to a region that extends upstream from the TATA sequence. TFIID is solely responsible for recognizing a promoter for RNA PolII. TFIID contains 2 types of components: the TATA-binding protein (TBP), a small protein of about 30 KDa, which is responsible for the recognition of the TATA box, and the so-called TAFs (for TBP-Associated Factors). Transcription factors act in a defined order to build a complex that is joined by RNA polymerase and is needed for the initiation of transcription. A promoter is initiated when TFIID binds the TATA box; then TFIIA joins the complex. The following step is the addition of TFIIB, which is bound downstream to the TATA box ; it may provide the surface that is recognized by RNA polymerase.The factor TFIIF consists of 2 subunits and it binds tightly to RNA PolII. TFIIF may bring RNA PolII to the assembling transcription complex. The initiation reaction, as defined by the formation of the first phosphodiester bond, can occur at this stage. Some further general transcription factors, TFIIE, TFIIH and TFIIJ, are required to allow RNA PolII to start moving away from the promoter. TFIIH has several activities, including an ATPase, a helicase, and a kinase activity that can phosphorylate and activate the RNA PoII; it is also involved in repair of

DNA damage. Most of the TFII factors are released before RNA PolII leaves the promoter. PROMOTERS AND ENHANCERS: Enhancers are short region of DNA that can increase transcription of genes Some transcription factors ("Enhancer-binding protein") bind to regions of DNA that are thousands of base pairs away from the gene they control. Binding increases the rate of transcription of the gene. Enhancers can be located upstream, downstream, or even within the gene they control. Enhancer-binding proteins in addition to their DNA-binding site, have sites that bind to transcription factors ("TF") assembled at the promoter of the gene. Differences between promoters and enhancers 1. A promoter must be immediately adjacent to the gene it controls. An enhancer can act from a long distance away. 2. A promoter has to be upstream of the gene it controls. An enhancer can act from upstream, downstream, or even within a gene. 3. A promoter can only act in one orientation. In other words, if a promoter is turned around 180 degrees, it will cease to function. An enhancer is functional in any orientation.

RIBOZYME:
A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule possessing a well defined tertiary structure that enables it to catalyze a chemical reaction. Ribozymes bind to the target RNA moiety through WatsonCrick base pairing and inactivate it by cleaving the phosphodiester backbone at a specific cutting site. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds (self-cleaving ribozymes), or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. Ribozymes in the laboratory that are capable of catalyzing their own synthesis under very specific conditions, such as an RNA polymerase ribozyme. Although most ribozymes are quite rare in the cell, their roles are sometimes essential to life. For example, the functional part of the ribosome, the molecular machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg2+ as cofactors. There is no requirement for divalent cations in a five-nucleotide RNA that can catalyze transphenylalanation of a four-nucleotide substrate which has three base complementary sequence with the catalyst. The ability of ribozymes to recognize and cut specific RNA molecules makes them exciting candidates for human therapy. Already, a synthetic ribozyme that destroys the mRNA encoding a receptor of Vascular Endothelial Growth Factor (VEGF) is being readied for clinical trials. Five classes of ribozymes have been described based on their unique characters in the sequences as well as three-dimensional structures (Bunnell,1997). They are denoted as (1) the Tetrahymena group I intron, (2) RNase P, (3) the hammerhead ribozyme, (4) the hairpin ribozyme, and (5) the hepatitis delta virus ribozyme. They may catalyze selfcleavage (intramolecular or "in-cis" catalysis) as well as the cleavage of external substrates.