Assignment no.2Bio302MC170404186QNO.1What is a pre-initiation complex? How a pre-initiation complex is formed?Answer:Pre-initiation complex: The complete set of generaltranscription factors and polymerase, bound together at the promoter andsuspended for initiation, is called the pre-initiation complex.Formationof pre-initiation complex:The generaltranscription factors help polymerase bind to the promoter and melt the DNA.They also help thepolymerase escape from the promoter and embark on the elongation phase. anypolymerase II promoters contain a TATAelement (which are 30 base pairs upstream of the transcription start site. Theformation of pre-initiation complex is start from this site.
The TATA elementis recognized by th general transcription factor called TFIID.TFII denotes thetranscription factor for polymerase II.Like many other general transcriptionfactors, TFIID is in fact, a multi subunit compound. The component of TFIIDthat binds to the TATA DNA sequence is called TBP (TATA binding protein).The other subunits in this complex arecalled TAFs, for TBP associated factors. Some TAFs recognize other corepromoter elements such as the initiator, DPE and DCE, although the strongestbinding is between TBP and TATA. Thus TFIID is a critical factor in promoterrecognition and establishment of pre-initiation complex.Upon binding DNA TBPextensively distorts the TATA sequence.
The resulting TBP DNA complex providesa platform to recruit other general transcription factors and polymerase itselfto the promoter In vitro these proteins assemble at the promoter in thefollowing order. First TFIIA, TFIIBthen TFIIF together with polymeraseand then TFIIE and TFIIH.The formation ofpre-initiation complex is followed by the promoter melting.(separation ofDNA strands). QNO2:What is RNA splicing? Describe the major events that occur in splicingpathway?Answer:RNA splicing:In this process removal of intron takesplace and RNA splicing convert the pre- mRNA into the mature mRNA that onlycontain exons.
RNA splicing must occur with great accuracyto avoid the loss or addition of even a single nucleotide at the site at whichthe exons are joined. Majorevents that occur in splicing pathwayIn a splicing pathway, a number of thesteps may differ slightly in their order or might even reverse.Initially the 5’splice site is recognized bythe U1 snRNP. U2AF is made up of 2 subunits the larger subunit is (65) whichbinds to the Py tract and the smaller is (35) binds to the 3’splice site. Theformer subunit interacts with BBP (SF1) and helps that protein bind to thebranch site.
This arrangement of protein and RNA is called early (E) complex.U2snRNP then binds to the branch site, aided by U2AF and displacing BBP(SF1).This arrangement is called the A complex.The base pairing between theU2 snRNPand the branch site A residue is extruded from the resulting stretch ofdouble-helical RNA as a single nucleotide bulge. This A residue is thus unpairedand available to react with the 5′ splice site. Thenext step is a rearrangement of the complex to bring together all three splicesite. This is achieved as follows, the U4 andU6snRNPs, along with the U5snRNPjoin the complex. Together these three snRNPs are called the tri- sn RNP particle.
With in this particle the U4 and U6 snRNP are held together by complementarybase pairing between their RNA components and the U5 snRNP is more looselyassociated through protein-protein interactions. With the entry of the tri-snRNP the A complex is converted into the B complex.In the next step, U1 leaves the complex U6replaces it at the 5’splice site.
This requires that the base pairing betweenthe U1 snRNP and the pre- mRNA be broken it is allowing the U6 RNA to annealwith the same region. Those steps complete the assembly pathway. Thenext rearrangement triggers catalysis and occurs as follows.U4 is released fromthe complex, allowing U6 to interactwith U2 through the RNA;RNA base pairing. This arrangement is called the Ccomplex which produces the active site.
That is the rearrangement bringstogether within the spliceosomethose component that together form the active site.The same arrangement also ensures that the substrate RNA is properly positionedto be acted upon. The formation of the active site juxtaposes the 5’splice siteof the pre- mRNA and the branch site facilitating the first transesterificationreaction. The second reaction between the 5’and 3’splice sitesis aided by theU5snRNP which help to bring the two exons together.The final step involves release of mRNA andsnRNPs. The snRNPs are initially still bound to the lariat but they getrecycled after rapid degradation of that piece of RNA.