TRANSCRIPTION--- SYNTHESIS OF RNA

1. TRANSCRIPTION---SYNTHESIS OF RNA Dr. Zakira Naureen

2. Previously we have studied • Central dogma of molecular biology • DNA Structure and Replication • Enzymes and factors involved in Replication • Three types of RNA m RNA; t RNA; r RNA

3. Learning objectives • What is gene ? • What is Transcription? • Structure of RNA polymerases

4. Genes • A gene is a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single mRNA which will be translated into a protein. • In eukaryotes, the genes have their coding sequences (exons) interrupted by non-coding sequences (introns). • In humans, genes constitute only about 2-3% of DNA, the rest is “junk”DNA.

5. Eukaryotic Gene Structure 5’ - Promoter Exon1 Intron1 Exon2 Terminator – 3’ UTR splice splice UTR transcription Poly A translation protein

6. Prokaryotic Gene Structure UTR UTR Promoter CDS Terminator Genomic DNA transcription mRNA translation protein

7. Prokaryote gene versus Eukaryote gene

8. What is Transcription ? • It is the process of copying DNA to RNA. • It differs from DNA synthesis in that only one strand of DNA, the template strand, is used to make mRNA. • Unlike DNA replication, it does not need a primer to start. • It can involve multiple RNA polymerases. • Transcription is divided into 3 stages • Initiation • Promoter recognition and binding • Initiation of polymerization of NTPs • Elongation • Termination

9. Transcription Tools • Template DNA • DNA-dependent RNA polymerase • ATP, GTP, CTP, and UTP are required, Mg2+ • Transcription factors • ATP

10. The coding strand and the template strand of DNA The important thing to realize is that the genetic information is carried on only one of the two strands of the DNA. This is known as the coding strand. The other strand is known as the template strand, and iscomplementaryto the coding strand. If you took the template strand and built a new DNA strand on it (as happens in DNA replication), you would get an exact copy of the original DNA coding strand formed. Almost exactly the same thing happens when you make RNA. If you build an RNA strand on the template strand, you will get a copy of the information on the DNA coding strand - but with one important difference.

11. The Code is read from 5’ to 3’

12. The Basics of Transcription

13. DNA Dependent RNA Polymerase • It’s a holoenzyme • Structure and function are fundamentally similar in Prokaryotes and Eukaryotes ( few exceptions) • Bacterial RNA Polymerases are composed of • 4 core subunits (2 small + 2 large ) + • Sigma factor (σ)– determines promoter specificity • Core + σ = holoenzyme • Binds promoter sequence

14. continued • In eukaryotes the DNA dependent RNA Polymerase has several other binding factors i.e. Transcription factors • More than one polymerases are required to make all different types of RNA unlike bacterial RNA Polymerase that can catalyze formation of all three RNA molecules. • Three polymerases for 3 RNA molecules • RNA pol I--------rRNA • RNA pol II-------mRNA • RNA pol III------tRNA

15. PROMOTER Sigma factors recognize consensus -10 and -35 sequences

16. continued • Promoter determines: • Which strand will serve as a template. • Transcription starting point. • Strength of polymerase binding. • Frequency of polymerase binding.

17. Prokaryotic Promoter • One type of RNA polymerase. • Pribnow box located at –10 (6-7bp) • –35 sequence located at -35 (6bp) • Sigma factor actually recognizes this and binds here

18. Eukaryote Promoter • Goldberg-Hogness or TATA located at –30 • Additional regions at –100 and at –200 • Possible distant regions acting as enhancers or silencers (even more than 50 kb).

19. Promoter Sequence

20. Promoter • Strong promoter resemble the consensus sequence. • Mutations at promoter sites can influence transcription. Human geneBeta globin

21. Stages of Transcription • Initiation – the RNA polymerase enzyme binds to a promoter site on the DNA and unzips the double helix. • Elongation – free nucleotides bind to their complementary pairs on the template strand of the DNA elongating the RNA chain which is identical to the informational strand of DNA, except that the nucleotide thymine in DNA is replaced by uracil in RNA. The polymerase moves along the DNA in the 3’ to 5’ direction, extending the RNA 5’ to 3’. • Termination – specific sequences in the DNA signal termination of transcription; when one of these is encountered by the polymerase, the RNA transcript is released from the DNA and the double helix can zip up again.

22. Transcription video

23. Chain Initiation • First phase of transcription is initiation • Initiation begins when RNA polymerase binds to promoter and forms closed complex • After this, DNA unwinds at promoter to form open complex, which is required for chain initiation

24. Initiation

25. Chain Elongation • After strands separated, transcription bubble of ~17 bp moves down the DNA sequence to be transcribed • RNA polymerase catalyzes formation of phosphodiester bonds between the incorp. ribonucleotides • Topoisomerases relax supercoils in front of and behind transcription bubble

26. Elongation

27. Chain Elongation (Cont’d)

28. Chain Termination • Two types of termination mechanisms: • intrinsic termination- controlled by specific sequences, termination sites • Termination sites characterized by two inverted repeats

29. Chain Termination (Cont’d) • Other type of termination involves rho () protein • Rho-dependent termination sequences cause hairpin loop to form

30. Transcription Regulation in Prokaryotes • In prokaryotes, transcription regulated by: • alternative s factors • enhancers • operons • transcription attenuation • Alternative s factors • Viruses and bacteria exert control over which genes are expressed by producing different s-subunits that direct the RNA polymerase to different genes.

31. Control by Different  Subunits

32. Enhancers • Certain genes include sequences upstream of extended promoter region • These genes for ribosomal production have 3 upstream sites, Fis sites • Class of DNA sequences that do this are called enhancers • Bound by proteins called transcription factors

33. Elements of a Bacterial Promoter

34. Operon • Operon: a group of operator, promoter, and structural genes that codes for proteins • the control sites, promoter, and operator genes are physically adjacent to the structural gene in the DNA • the regulatory gene can be quite far from the operon • operons are usually not transcribed all the time • b-Galactosidase, an inducible protein • coded for by a structural gene, lacZ • structural gene lacY codes for lactose permease • structural gene lacA codes for transacetylase • expression of these three structural genes is controlled by the regulatory gene lacI that codes for a repressor

35. How Does Repression Work • Repressor protein made by lacI gene forms tetramer when it is translated • Repressor protein then binds to operator portion of operon • Operator and promoter together are the control sites

36. Binding Sites On the lac operon • Lac operon is induced when E. coli has lactose as the carbon source • Lac protein synthesis repressed by glucose (catabolite repression) • E. coli recognizes presence of glucose by promoter as it has 2 regions: RNA polymerase binding site, catabolite activator protein (CAP) binding site

37. Binding Sites On lac operon (Cont’d)

38. Catabolite Repression • CAP forms complex with cAMP • Complex binds at CAP site • RNA polymerase binds at available binding site, and transcription occurs

39. Basic Control Mechanisms in Gene Control • Control may be inducible or repressive, and these may be negatively or positively controlled

40. Control of the trp operon • Trp operon codes for a leader sequence (trpL) and five polypeptides • The five proteins make up 4 different enzymes that catalyze the multistep process that converts chorisimate to tryptophan

41. Alternative 2˚ structures Can Form in trp Operon • These structures can form in the leader sequence • Pause structure- binding between regions 1 and 2 • Terminator loop- binding between regions 3 and 4 • Antiterminator structure- Alternative binding between regions 2 and 3

42. Attenuation in the trp operon • Pause structure forms when ribosome passes over Trp codons when Trp levels are high • Ribosome stalls at the Trp codon when trp levels are low and antiterminator loop forms

43. Transcription in Eukaryotes • Three RNA polymerases are known; each transcribes a different set of genes and recognizes a different set of promoters: • RNA Polymerase I- found in the nucleolus and synthesizes precursors of most rRNAs • RNA Polymerase II- found in the nucleoplasm and synthesizes mRNA precursors • RNA Polymerase III- found in the nucleoplasm and synthesizes tRNAs, other RNA molecules involved in mRNA processing and protein transport

44. RNA Polymerase II • Most studied on the polymerases • Consists of 12 subunits • RPB- RNA Polymerase B

45. How does Pol II Recognize the Correct DNA? • Four elements of the Pol II promoter allow for this phenomenon

46. Initiation of Transcription • Any protein regulator of transcription that is not itself a subunit of Pol II is a transcription factor • Initiation begins by forming the preinitiation complex • Transcription control is based here

47. General Transcription Initiation Factors

48. Transcription Order of Events • Less is known about eukaryotes than prokaryotes • The phosphorylated Pol II synthesizes RNA and leaves the promoter region behind • GTFs are left at the promoter or dissociate from Pol II

49. Elongation and Termination • Elongation is controlled by: • pause sites, where RNA Pol will hesitate • anti-termination, which proceeds past the normal termination point • positive transcription elongation factor (P-TEF) and negative transcription elongation factor (N-TEF) • Termination • begins by stopping RNA Pol; the eukaryotic consensus sequence for termination is AAUAAA

50. Gene Regulation • Enhancers and silencers- regulatory sequences that augment or diminish transcription, respectively • DNA looping brings enhancers into contact with transcription factors and polymerase