FOB 9 transcription

• RNA vs DNA


  second carbon has hydroxyl group, RNA has uracil instead of thyamine.
• non template strand or


  coding strand. actually has the code of the rna
• template strand or


  non coding strand, acts as a template for the reverse compliment therefore not the code
• general steps to complete rna


  5 prime cap to prevent degradation (phosphate group to another phosphate group) 3 prime poly/tail 80-150 A nuceltides on 3 prime tail. splice out introns (non coding sequences, regulatory) so only exons are left (coding sequence) phosphodiester bonds are cut then re-fused
• genome


  entirtely of hereditary genetic information of a cell
• transcriptome


  sum of all RNA molecules produced in a cell at any given time. shows wich gene are active vs silent
• transcription


  the process of creating a complementary RNA copy of a sequence of DNA
• translation


  the process by which an mRNA sequence is decoded (converted) into a specific amino acid sequence in the construction of a polypeptide chain
• transcription vs replication


  similarites: one strand is a template, 5' to 3'. 3 phases (initiation, elongation, termination). differences: only one strand acts as a template, no primers are needed, only small areas of DNA are used as a template (gene/genetic region) and newly synthesised strand is RNA not DNA (T to U, 2nd carbon on ribose has hydroxyl)
• start sequence for transcription terms


  5' to 3' of starting sequence for transcription is 'downstream and given a positive number +1, +2 etc). 3' to 5' is 'upstream' and given negative number
• template strands in genome


  can be any gene or part of a gene in top and bottom strands.
• promoter genes transcription

  polymerase binding occurs within the promoter of a gene. a specific sequence of DNA, e.coli typically spans a region of -70 to 30+ bp. and contains consensus sequences with different organisms. 10bp 5'-TATAAT-3' Tata box, 35bp 5'-TTGACA-3', 40 to 60 pb AT rich region (upstream promoter UP for highly expressed genes). highly conserved so polymerase knows were to bind. the spacing is particularly important, as this is associated with RNA polymerase binding
• initiation polymerase


  2 a, 2b and one w Omega subunit. has its own helicase and gyrase activity (splits strands and prevents torsional strain). sigma subunit binds temporarily to core subunits and guides polymerase to promoter sequences, each subunit is specific to certain genes, sigma 70 is common. no proofreading, higher mistake rate 10^4 to 10^5. this is not as crucial as mRNA is not long-lived and there are many copies. also not passed onto proginey cells. transcription occurs faster than replication
• polymerase binding process


  binds to template strand using two magnesium co-factors. nucleotide triphophates are brought in and added in. nucleophilic attach through free 3 prime hydroxyl group of previous nucleotide and alpha phosphate group from the NTP. forming a phosphodiester bond, Mg 1 stabilises this attack, Mg 2 holds to remaining two phosphates which are cleaved and used as energy for the reaction. no primer helicate or DNA binding proteins are needed.
• initiation transcription


  polymerase driven by sigma subunit to promoter region. binds causing a closed complex until internal helicase activity splots open 12-15 bp of DNA forming open complex. elongation involves promoter clearance, and the sigma subunit dissociates once the DNA is out of the promoter region
• elongation


  begun movememnt along template strand and sigma subunit dissociated. 5-3 adding to OH. NTP not dNTP. polymerase unwinds 17bp per time and rewind after. an 8bp DNA-RNA hybrid double helix forms at any one time before RNA dissociates and DNA rewinds. polymerase moves 50-90 nucleotides/sec. Topoisomerase prevents supercoiling.
• termination


  Rho dependant or independent. polymerase reafches terminator sequences where it pauses, and complex is broken down. dependant - rut site has rho protein which binds and runs along rna. when polymerase pauses roh protein reaches polymerase and causes the complex to break rna from DNA and causing polymerase to dissociate. independent - just before pause site is complimentary region, allows the rna to form a hairpin/stem loop complex which puts pressure on RNA-DNA connection. a line of AAA is present after which is easily broken, polymerase then dissociates
• eukoryotic vs prokaryotic transcription


  general process is the same, eukarotic is mroe complex. prokaryotic has 3 polymerases. 1 - produces pre- (ribosomal) rRNA (18S, 5.8S and 28S). 2- produces mRNA and specialised RNAs, large variety of promoters recognised by pol 2. still some common consensus sequences e.g. TATA box and initiator sequences +1bp. but polymerase recognises many. RNA pol II produces tRNA, 5S rRNA and specialised RNAs
• RNA pol II


  contains 12 subunits RPB1-12. RPB1 contains a long carboxyl-terminal domain CTD separate from the main body that is important for Pol II functioning (does 5' cap and 3' tail). very complex due to genome complexity and its packaging/association with numerous proteins. requires numerous numerous transcription factors for proper functioning (formation of an active transcription complex). naming factors for Pol II: TFII followed by an addition idenitfer e.g. TFIIF. There are general TF always present, then specialised ones for certain genes
• polymerase II initiation


  closed complex formation initiated by TATA binding protein binding TBP to the TATA box. no box: TBP arrives part of complex TFII(2)D. both TFIIA n TFIIB bind TBP or TFIIB stabilising interaction dsDNA. A forms attachment point for b which gives connection point and signals for Pol II attachment which has TFIIF (helps to target promoter) bound. TFIIE and TFIIH (helicase activity, unwinds 12-15 N at promoter, opening complex) phosphorylate pol II CTD tail activating complex inducing transcription. synthesis of 1st 60-70 nucleotides TFIIE and TFIIH are released.
• transcription facotrs


  bind and regulation transcription, can be always present in polymerase binding, or some are specific to particular genes. regulate rate and isoforms of genes. generic or specific. in prokaryotic system
• polymerase II elongation and termination


  TFIIF remains bound to pol 2 during elongation. polymerisation is enhanced by binding of elongation factors (important in preventing pausing of transcription and in allowing post-transcriptional modification in association with CTD) which are replaced by termination factors. once elongation has terminated Pol II is phosphorylated and recycled.
• polymerase 2


  involved in initiation, catalyzes RNA synthesis
• TBP (tata binding protein)


  specifically recognizes the TATA box
• TFIIA


  stabilizes the binding og TFIIB and TBP to the promoter
• TFIIB


  inds to TBP and recruits the Pol II tfIIf complex
• TFIID


  required for initiation at promotere lacking a tata box
• TFIIE


  binds tihgtly to pol II, binds to TFIIB and prevents binding of Pol II to nonspecific DNA sequences
• TFIIH


  unwinds DNA at promoter (helicase activity), phosphorylates pol II within the CTD, recruits nucleotide excision repair proteins.
• RNA processing (post-transcriptional modifications)


  eukaryote only. 5' capping (addition of 7-methyl guanosine to 5' terminal), 3' poly(A) tail (addition of 80-259 A to 3' terminal mRNA), splicing (removal of introns, can be self-splicing or require snRNPs)
• 5' capping


  the first RNA base has 3 phosphates still attached as nothing is linked to it, one is given off and 7-methylguanosine is added as usual (2 of 3 phosphates cleaved for energy) forming 5',5' triphosphate linkage. this abnormal structure (including methyl group on G and occasionally 2nd carbon of 1st n 2nd ribose) signal it is mRNA and thus protects against ribonucleases and act as recognition sequence.
• poly A tail


  occurs at CTD. transcription is extended beyond the site where the poly A tail will be added. cleavage of RNA and addition of Poly A tail is marked by recognition sequence (AAUAAA). polyadenylation factors bound to CDT tail, downstream of recognition sequence endonuclease cleaves off the rest of sequence after recognition. polyadenylate polymerase adds of 80-50 A residues. acts as recognition sequence in translation and helps protect against enzymatic degradation
• splicing


  introns are regulatory and noncoding therefore they need to be removed. self-splicing (no ezymes, group 1 and 2). require spliceosomes (more common, group 3 and 4, for mRNA. for more complex and bigger introns)
• self splicing

  1:exonU to A intron (common). free-floating guanosine nucleotide which can undertake nucleophilic attack between U and A. leaves a free 3' hydroxyl group on exon which attacks the other exon. requires a guanine nucleotide. 2: nucleotide in pre-existing intronic region, free 2' hydroxyl group undergoes a nucleophilic attack splicing 1st intron exon connection, forms lariat. 3' oh group on exon attacks other exon. requires a 2'OH group of an A in the intron which is degraded n reused. both have two trans-esterification reactions and formation of new phosphodiester bonds
• spliceosome


  specialised RNA-protein complex. small nuclear ribonucleoprotein (snRNP) 'snurps'. each snurp contains one class of RNA 110-200 nucleotides long, small nuclear rna (snRNAs). 5 major snRNA involved in splicing (U1,2,4,5 n 6) spliceosomal introns normally have the sequence GU at 5' n AG at 3'. U1 binds GU and u2 binds AG end. U2 contains A nucleotide for nucleophilic attack. U4, U6 and u5 are bound forming spliceosome. ATP energy is required to form the spliceosome, splicing method is similar to group 2. U1 and U4 are removed and A cleaves GU creating lariat and exon attacks other
• order of rna processing


  5' cap at beginning of transcription, 3' tail at the end. splicing as final process
• alternative splicing


  multiple RNA products from one gene, depending on how it is spliced. contains at least 2 poly A sites which change what exons are included. exons can also be skipped
• viral replication


  RNA to DNA, catalysed by reverse transcriptase. virus forms reverse compliment DNA strand to the RNA template strand, RNA-DNA complex is degraded and replaced with 2 DNA strands. this is added to the host's genome and proteins can be produced. reverse transcriptase has high error rate, leading to high mutation rate and faster viral evolution. some viruses also creat RNA from RNA wit ran dependant RNA polymerase