TRANSCRIPTION Dr. Harinikumar. K.M. University of Agricultural sciences Bengaluru Karnataka

1. TRANSCRIPTIONDr. Harinikumar. K.M.University of Agricultural sciences Bengaluru Karnataka www.powerpointpresentationon.blogspot.com

2. The Central Dogma The central dogma of molecular biology was first articulated by Francis Crick in 1958 and re-stated in a Nature paper published in 1970 The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid. In other words, the process of producing proteins is irreversible: a protein cannot be used to create DNA.

3. Prokaryotes : Relatively Simple….

4. Eukaryotes : Not so Simple….

5. Introduction • The majority of genes are expressed as the proteins they encode. The process occurs in two steps: • Transcription = DNA → RNA • Translation = RNA → protein

6. Terminology Related To Transcription • Coding strand (or sense strand or non-template strand)-The DNA strand that has the same sequence as the mRNA and is related by the genetic code to the protein sequence that it represents. • RNA polymerase – An enzyme that synthesizes RNA using a DNA template (formally described as a DNA-dependent RNA polymerase). FIGURE :Promoters and terminators define the unit

7. Promoter –A region of DNA where RNA polymerase binds to initiate transcription. • Start point –The position on DNA corresponding to the first base incorporated into RNA. • Terminator –A sequence of DNA that causes RNA polymerase to terminate transcription. • Transcription unit – The sequence between sites of initiation and termination by RNA polymerase; it may include more than one gene.

8. Upstream –Sequences in the opposite direction from expression. • Downstream –Sequences proceeding farther in the direction of expression within the transcription unit. • Primary transcript –The original unmodified RNA product corresponding to a transcription unit.

9. Types of RNAs Produced in Cells

10. Phases of Transcription • Initiation:Binding of RNA polymerase to promoter, unwinding of DNA, formation of primer. • Elongation:RNA polymerase catalyzes the processive elongation of RNA chain, while unwinding and rewinding DNA strand • Termination:termination of transcription and disassemble of transcription complex.

11. RNA Polymerase Structure: Theme and Variation on Five Subunits

12. Four RNA Polymerases of Eukaryotic Cells

13. Initiation RNA pol I RNA pol III RNA pol II __________________________________________________ ATP requirement no no yes __________________________________________________ A and B or TATA box core consensus sq. core element C box Inr __________________________________________________ CAAT box upstream element UCE GC box etc __________________________________________________ general TFs SL1 TFIIIA B C various TFIIs ___________________________________________________ upstream factors UBF various up- stream factors _____________________________________________________

15. Transcription Initiation by RNA Polymerase II • Initiation occurs at an initiation site at the 3’ end of the gene. The initiation site is part of a larger promoter. Each gene has its own promoter. The promoter consists of a TATA box of about 100 nucleotides, mostly T and A, and an initiation sequence. The TATA box is upstream of the initiation sequence. • Proteinaceous transcription factors attach to the promoter and help the polymerase find and attach to the initiation site. RNA polymerase attaches to the initiation sequence.

16. Bacterial RNA Polymerase Consists of Multiple Subunits • Holoenzyme – The RNA polymerase form that is competent to initiate transcription. It consists of the five subunits of the core enzyme and σ factor. • Bacterial RNA core polymerases are ~400 kDmultisubunit complexes with the general structure α2ββ′. Catalysis derives from the β and β′ subunits. • CTD (C-terminal domain) – The domain of RNA polymerase that is involved in stimulating transcription by contact with regulatory proteins. FIGURE : RNA polymerase has 4 types of subunit

17. RNA-pol of E. Coli Rifampicin, a therapeutic drug for tuberculosis treatment, can bind specifically to the  subunit of RNA-pol, and inhibit the RNA synthesis.

18. E. Coli RNA Polymerase • Bacterial RNA polymerase can be divided into the α2ββ′ core enzyme that catalyzes transcription and the  subunit that is required only for initiation. • The β’ subunit is involved in DNA binding. • The β subunit contains the polymerase active site. • The α subunit acts as scaffold on which the other subunits assemble. Site of DNA binding and RNA polymerization

19. Sigma factor • Also requires sigma factor for initiation –forms holo enzyme complex • Sigma factor changes the DNA-binding properties of RNA polymerase so that its affinity for general DNA is reduced and its affinity for promoters is increased. • The Sigma factor is required for binding of the RNA polymerase to the promoter • Association of the RNA polynerase core complex omega the Sigma factor forms the holo-RNA polymerase complex • omega the Sigma factor the core complex binds to DNA non-specifically. FIGURE : Sigma factor controls specificity

20. Sigma factors • decreases the affinity of the RNA polymerase to non-promoter regions • Different Sigma factors for specific classes of genes • Different sigma factors bind RNAP and recognize specific -10 ,-35 sequences • Helps melt DNA to expose transcriptional start site • Most bacteria have major and alternate sigma factors • Promote broad changes in gene expression • E. coli 7 sigma factors • B. subtilis 18 sigma factors Generally, bacteria that live in more varied environments have more sigma factors

21. Sigma factors s70 s54 sS sS sF s32 Extreme heat shock, unfolded proteins E. coli can choose between 7 sigma factors and about 350 transcription factors to fine tune its transcriptional output

22. The Holoenzyme Goes through Transitions in the Process of Recognizing and Escaping from Promoters • When RNA polymerase binds to a promoter, it separates the DNA strands to form a transcription bubble and incorporates nucleotides into RNA. FIGURE :- RNA polymerase surrounds the bubble

23. Transcription Occurs by Base Pairing in a “Bubble” of Unpaired DNA • RNA polymerase separates the two strands of DNA in a transient “bubble” and uses one strand as a template to direct synthesis of a complementary sequence of RNA. • The bubble is 12 to 14 bp, and the RNA–DNA hybrid within the bubble is 8 to 9 bp. • RNA-polrecognizesthe TTGACA region, and slides to the TATAAT region, then opens the DNA duplex. • The unwound region is about 171bp. • No primer is needed for RNA synthesis FIGURE RNA synthesis occurs in the transcription bubble

24. FIGURE :- RNA polymerase passes through several steps prior to elongation

25. Ternary complex – The complex in initiation of transcription that consists of RNA polymerase and DNA as well as a dinucleotide that represents the first two bases in the RNA product. • There may be a cycle of abortive initiations before the enzyme moves to the next phase. • Sigma factor is usually released from RNA polymerase when the nascent RNA chain reaches ~10 bases in length.

26. FIGURE:- RNA polymerase changes size at initiation

27. Sigma Factor Controls Binding to DNA by Recognizing Specific Sequences in Promoters • conserved sequence – Sequences in which many examples of a particular nucleic acid or protein are compared and the same individual bases or amino acids are always found at particular locations. • A promoter is defined by the presence of short consensus sequences at specific locations. • The promoter consensus sequences usually consist of a purine at the startpoint, a hexamer with a sequence close to TATAAT centered at ~ –10(–10 element or TATA box), and another hexamer with a sequence similar to TTGACA centered at ~–35(–35 element).

28. Individual promoters usually differ from the consensus at one or more positions. • Promoter efficiency can be affected by additional elements as well. • UP element – A sequence in bacteria adjacent to the promoter, upstream of the –35 element, that enhances transcription. FIGURE 14: DNA elements and RNA polymerase modules contributing to promoter recognition by sigma factor

29. Promoter Efficiencies Can Be Increased or Decreased by Mutation • Down mutationsto decrease promoter efficiency usually decrease conformance to the consensus sequences, whereas up mutations have the opposite effect. • Mutations in the –35 sequence can affect initial binding of RNA polymerase. • Mutations in the –10 sequence can affect binding or the melting reaction that converts a closed to an open complex.

30. Transcription Factors • TFAIIA, TFAIIB – components of RNA polymerase II holo-enzyme complex • TFIID – Initiation factor, contains TATA binding protein (TBP) subunit. TATA box recognition. • TFIIF – (RAP30/74) decrease affinity to non-promoter DNA

31. Class II transcription factors • TFIIA activates TBP by relieving the repression that is caused by the TAFs • TFIIB binds adjacent to TBP and TATA box • TFIID is a complex protein containing a TATA-box binding protein and 8-10 TBP-associated factors (TAFs) • TBP:TATA-binding protein • TAFs:TBP-associated factors • TFIIF consists of two subunits. The larger subunit has an ATP-dependent DNA helicase activity and the small one contacts the core polymerase. • TFIIE and TFIIH are required for promoter clearance to allow RNA polymerase to commence movement away from the promoter.

32. TF for eukaryotic transcription

33. polⅡ TFⅡF TFⅡH TAF TAF TAF TFⅡA TBP TFⅡB DNA TATA RNA polⅡ with transcription factors form transcription initiation complex. TF II D is the only factor which can recognize specific sites.

34. POL-Ⅱ POL-Ⅱ TFⅡF CTD- P Pre initiation complex ⅡB TBP TBP TBP TBP TAF TAF TAF TATA ⅡA TFⅡD-ⅡA-ⅡB-DNA complex CTD（Carboxyl Terminal Domain ) is repeated sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser ⅡH ⅡE ⅡH ⅡE TFⅡF TBP TAF ⅡB TATA ⅡA CTD tail of RNA pol II is phosphorylated by TFⅡH

35. Pre-initiation complex (PIC) • TBP of TFII D binds TATA • TFII A and TFII B bind TFII D • TFII F-RNA-pol complex binds TFII B • TFII F and TFII Eopen the dsDNA (helicase and ATPase) • TFII H: completion of PIC

41. Model of RNA PolII Preinitiation Complex

43. Interactions between Sigma Factor and Core RNA Polymerase Change During Promoter Escape • A domain in sigma occupies the RNA exit channel and must be displaced to accommodate RNA synthesis. • Abortive initiations usually occur before the enzyme forms a true elongation complex. • Sigma factor is usually released from RNA polymerase by the time the nascent RNA chain reaches ~10 nt in length. FIGURE :-Sigma and core enzyme must dissociate

44. Enzyme Movement • DNA moves through a channel in RNA polymerase and makes a sharp turn at the active site. • Changes in the conformations of certain flexible modules within the enzyme control the entry of nucleotides to the active site. FIGURE :- DNA turns at the active site

45. Elongation: • RNA polymerase moves along, unwinding one turn of the double helix at a time thus exposing about 10 bases. • New RNA nucleotides are added to the 3’ end of the growing mRNA molecule at a rate of about 60 sec-1. • The double helix reforms behind the enzyme. Many RNA polymerase molecules can transcribe simultaneously (remember, the gene is hundreds of thousands of nucleotides long). • Only one DNA strand, the coding strand, is transcribed. The other strand is not used (silent). But, which strand is coding and which silent varies from gene to gene. • The reading direction is 3’ to 5’ along the coding strand.

46. Polymerase is accurate - only about 1 error in 10,000 bases • Even this error rate is OK, since many transcripts are made from each gene • Elongation rate is 20-50 bases per second - slower in G/C-rich regions • Topoisomerases precede and follow polymerase to relieve supercoiling

47. Termination: • Process by which RNA polymerase complex disassembles from 3’ end of gene. • There are two classes of terminators: Those recognized solely by RNA polymerase itself without the requirement for any cellular factors are usually referred to as “intrinsic terminators.” • Others require a cellular protein called rho and are referred to as “rho-dependent terminators.”

48. 1. Rho-independent termination • Termination sequence has 2 features: • Series of U residues • GC-rich self-complimenting region • GC-rich sequences bind forming stem-loop • Stem-loop causes RNAP to pause • U residues unstable, permit release of RNA chain

49. 2. Rho-independent termination • Rho factor is a protein that binds to nascent RNA and tracks along the RNA to interact with RNA polymerase and release it from the elongation complex. • rut – An acronym for rho utilization site, the sequence of RNA that is recognized by the rho termination factor. • Rho is hexameric protein • 70-80 base segment of RNA wraps around • Rho has ATPase activity, moves along RNA until site of RNAP, unwinds DNA/RNA hybrid • Termination seems to depend on Rho’s ability to “catch up” to RNAP • No obvious sequence similarities, relatively rare

50. FIGURE :- Rho terminates transcription