Gene Expression (Transcription) PDF

IMB 37 events of cell division. Gene Expression (Transcription) 1) Transcription in Prokaryotes - Step 1: Initiation Initiation in prokaryotes: - There is only (1) RNA polymerase for sy...

IMB 37 events of cell division. Gene Expression (Transcription) 1) Transcription in Prokaryotes - Step 1: Initiation Initiation in prokaryotes: - There is only (1) RNA polymerase for synthesis of 3 types RNA. Prokaryote RNA polymerase is formed of sigma factor and 2α and 2β subunits and Sigma factor(σ) it recognizes and binds the promoter at TATA box then RNA polymerase binds the start point. - Antibiotic (Rifampicin) binds β subunitand inhibits RNA synthesis. - There are two boxes for identification of initiation of transcription in prokaryotes: 1. TATA Box: it is a specific sequence of nucleotides located 10 bases upstream of start 2. TTGACA BOX : it is a specific sequence of nucleotides located 35 bases upstream of start point. it determines frequency of transcription. IMB 38 - Step 2: Elongation - RNA polymerase creates +ve supercoil in front of its movement and –ve supercoil behind it. - Gyrase enzyme release +ve supercoil in front of RNA polymerase. - Topoisomerase enzyme release – ve supercoil behind RNA polymerase. - Step 3: Termination - Rho factor dependant: a specific termination protein. - The simplest stop signal is a palindromic GC region immediately followed by a T-rich region. The palindromic GC-rich region forms a hairpin structure in the RNA due to the base pairing. - There are also proteins that assist in terminating RNA transcription - One such protein discovered in prokaryotes is the rho ( ρ) protein This binds to the newly made RNA at C-rich G-poor regions and scans along the RNA towards the RNA polymerase in an ATP- dependent manner When it catches up with the RNA polymerase, it breaks the DNA/ RNA association and thus terminates transcription. - Rho independent RNA forms hairpin preventing attachment of RNA polymerase. In absence of hair pin loop another mechanism involves the presence of (Histone mRNAs ) that are processed by a histone- specific mechanism to end after a highly conserved IMB 39 RNA hairpin element. - Transcription bubble is formed of template DNA ,RNA polymerase, newly formed RNA 2) Eukaryote Transcription - There are 3 polymerases in eukaryotes - RNA polymerase I for synthesis of r RNA - RNA polymerase II for synthesis of m RNA - RNA polymerase III for synthesis of t RNA - Step 1: - Preinitiation complex: RNA polymerase II is composed of 12 subunits and is the target of regulation by multiple transcription factors that specify which genes are transcribed. - The promoter is the binding site for transcription factors that form a preinitiation complex (PIC). - RNA polymerase II does not bind directly to the promoter sequence but to the PIC. https://www.slideshare.net/adurganaveen/transcription-57863890 IMB 40 - Step 2: Initiation - TATA box is present-25 bases upstream from initiation of transcription it determine site of initiation of transcription. The first step is the binding of the TATA box-binding protein (TBP) and TBP-associated factors (TAFs, collectively known as TFIID) to the TATA box. - CAAT box (CCAATC) is present 40 bases from initiation of transcription. - GC box (GGCGGG) is present 200 bases from initiation of transcription ( determine frequency of transcription. - Transcription factor IID (TFIID) - The first step is the binding of the TATA box-binding protein (TBP) and TBP-associated factors (TAFs, collectively known as TFIID) to the TATA box. - Then recruitment of other transcription subunits namely TFIIA, TFIIB, TFIIF This is followed by RNA polymerase II itself and, finally, TFIIE and TFIIH - Unwinding of a short stretch of the DNA double helix to reveal the single-stranded that it will be transcribed.The rate of transcription initiation by the the end of this process is named as : Basal Transcription Apparatus - Enhancer sequences can, when bound, also modify the rate of initiation complex formation. The rate of transcription is controlled by the stability of the com plex, which can dissociate easily from the promoter - Transcription starts by phosphorylation of the enzyme by kinase. 3) Elongation RNA synthesis requires unwinding of DNA then the enzyme RNA polymerase forms new RNA strand. 4) Termination IMB 41 - The “end” of the newly formed RNA occurs between AAUAAA sequence followed by GU-rich sequence separated by about 40-60 nucleotides in the emerging RNA. Once both of these sequences have been transcribed, a protein called Polyadenylation Specificity Factor (CPSF) in humans binds the AAUAAA sequence and a protein called Cleavage Stimulation Factor ( CstF ) in humans binds the GU-rich sequence. - The Poly(A) Polymerase enzyme which catalyzes the addition of a 3′ poly-A tail on the pre-mRNA is part of the complex that forms with CPSF and CstF. Post transcriptional Modification of mRNA Processing of mRNA - Capping - A 7-methylguanosine is linked to the first transcribed nucleotide through a 5'-5' triphosphate bridge to form a 5' methyl cap. After this occurs by a capping enzyme and an RNA (guanine-7-) methyltransferase. - Capping is important for 1) Protection of 5 end from exonuclease. 2) 5' methyl cap binds to a translation factor (eIF4E), which is the first step of mRNA recruitment to the 40S ribosome subunit. - Polyadenylation - It is the process of addition of poly adenine nucleotides to the end of mRNA( tail) at its 3 end. Polyadenylation occurs during and/or immediately after transcription of DNA into RNA. It is about 100-200 bases of Adenine bases. (This reaction is catalyzed by polyadenylate polymerase) - Polyadenylation is important for: 1) Protection of 3 end from exonuclease. IMB 42 2) It is important for transcription termination, export of the mRNA from the nucleus and translation. - RNA Splicing - It means excision of interons and joining of exon ends. It requires small nuclear RNA (splicesomes) this process is important for gene rearrangement coding for different proteins. - Five specific snRNAs are essential for binding to more than 100 non-small nuclear ribo- nuclear protein-splicing factors to form small nuclear ribonucleo- proteins (snRNPs). - Assembling and capping of snRNAs with trimethylguanosine to be different from other mRNAs. - Adding of several uridine-rich proteins (U1, U2, U4, U5, and U6), to the cap to reenter the nucleus. - Alternative splicing: An extra level of complexity is added to splicing by alternative splicing in which an mRNA can be spliced differently, thereby leading to a different assortment Processing of t RNA - It has a clover leaf appearance and it is synthesized by RNA polymerase III it processed by : 1) Removal of extension at 5 2) Adding of CCA at 3︡ 3) Excision of interons at anticodon 4) Modification of some bases by methylation of Uracil Processing of r RNA - It is formed by RNA Polymerase I and 5s by RNA polymerase III. - It undergoes processing and cleavage in the nucleus into 28S,5.8S and 18S. IMB 43 - Comparison between Transcription in Prokaryote and Eukaryote Prokaryote eukaryote One enzyme formed of 2α & 2β and 3 enzymes RNA polymerases I &II &III enzyme sigma factor TATA Box and TTGACA box Initiation TATA &CAAT & GC Boxes located located upstream of start upstream of initiation Sigma factor recognize promoter at TFII D binds the TATA box at site of TATA and binds it initiation Gyrase enzyme release +ve RNA polymerase binds with TFIID forming supercoil in front of RNA (pre-initiation complex). Elongation polymerase RNA synthesis; unwinding of DNA then Topoisomerase enzyme release – RNA polymerase forms new RNA strand ve supercoil behind RNA polymerase Rho factor dependant specific Specific sequence (AAUAAA)appears in Termination termination protein RNA then RNA polymerase is dissociated Rho independent RNA forms hairpin and dephosphorylated preventing attachment of RNA polymerase.