transcription

1. "The Central Dogmaof molecular biology" transcription translation replication DNA RNA protein Reverse transcription

3. Chapter 10 Transcription (RNA Biosynthesis)

5. RNA DNA Transcription*: RNA biosynthesis from a DNA template is called transcription. transcription • products：mRNA tRNA rRNA

7. Enzymes and Proteins involved in transcription ： • substrates : NTP • ( ATP, UTP, GTP, CTP ) • template: DNA • enzyme : RNA polymerase • the other Protein factors

8. Chemical reaction-- polymerization reaction: RNA polymerase catalyze formation of Phosphodiester bonds and release pyrophosphate (ppi) RNA polymerase RNA precursor

9. RNA biosynthesis is similar to DNA biosynthesis*: • Template- DNA • Enzyme—dependent on DNA • Chemical reaction--the formation of Phosphodiester bonds • Direction of synthesis--- 5’ 3’ • obey the ruler of base paired

10. RNA biosynthesis includes three stages: • Initiation: RNA polymerase binds to the promoter of DNA, and then a transcription “bubble” is formed. • Elongation: the polymerase catalyzes formation of 3’5’-phosphodiester bonds in 5’3’ direction, using NTP as building units. • Termination: when the polymerase reaches a termination sequence on DNA, the reaction stops and the newly synthesized RNA is released.

11. Formation of a transcription bubble

12. 1. RNA biosynthesis in prokaryotes

13. RNA polymerase in E. coli : consists of five subunits, a2bb’ws, which is called “holoenzyme”. The s subunit functions as a starting factor that can recognize and bind to the promoter site. The rest of the enzyme, a2bb’w, is known as “core enzyme”, responsible for elongation of the RNA sequence.

15. 53 35 RNA-pol • Important terms in RNA biosynthesis. • Operon*: a coordinated unit of gene expression, • which usually contains a regulator gene and a set • of structural genes. • B) Promoter site*: a region of DNA templates that • specifically binds RNA polymerase and determines • where transcription begins. Promoter site regulator gene structural genes

16. The –10 sequence: refers to the consensus TATAAT, and is known as “Pribnow box”. • The –35 sequence: refers to the consensus TTGACA, which is recognized by the s subunit of RNA polymerase, recognition site

17. 5  3  3  5  -5 0 -40 -30 -20 -10 1 10 -35 -10 T T G A C A A A C T G T T A T A A T Pu A T A T T A Py (Pribnow box) the site of transcription (the start site) consensus sequences region region recognition site

18. C) Sense and antisense strand: The antisense (-) strand refers to the DNA strand that is used as template to synthesize mRNA. The sense (+) strand of a DNA double helix is the non-template strand that has the same sequence as that of the RNA transcript except for T in place of U. Antisense (-) strand = template strand Sense (+) strand = coding strand

19. coding strand 5 3 3 5 template strand Sense and antisense strand: antisense strand sense strand structural gene

21. 3) Process of RNA biosynthesis: The process is similar to DNA synthesis but no primer is needed and T is replaced by U. • Initiation: • σ factor recognizes the initiation site（-35 region）, the • holoenzyme of RNA-pol bind to duplex DNA and move • along the double helix towards –10 region. • the holoenzyme of RNA-pol arrived on –10 region，and • bind to –10 region ，DNA is partially unwound and was • opened 10-20 bp length.

23.  ppi • Then incoming 2 neighbour nucleotides which base pairs are complementary with DNA template, RNA polymerase catalyzed the first polymerization reaction. 5’-pppG -OH + NTP –5’ -pppGpN – OH + ppi pppG pppGpN - OH NTP initiation complex: RNApol(α2ββˊσ)-DNA-pppGpN-OH3’

25. B) Elongation: after the first phosphodiester bond has been formed, the s subunit is released. The core enzyme moves in a 5’3’ direction on the DNA strand while it is catalyzing elongation of the RNA transcript.

26. RNA-pol (core enzyme)····DNA····RNA tanscription complex:

29. C) Termination: when the core enzyme reaches a termination sequence, the region near the 3’end of RNA forms a hairpin structure by self base-pairing. The transcription stops, the core enzyme and the newly synthesized RNA are released. For those DNA templates that lack the sequence to produce a hairpin structure of the RNA transcript, a protein factor called “r ” recognizes the termination site, stops transcription, and causes release of the newly synthesized RNA.

30. A hairpin structure at the 3’end of RNA

33. Subunits of RNA polymerase in E. coli • Subunit Size (AA) Function • 329 required for assembly of the enzyme; interacts with some regulatory proteins; involved in catalysis • 1342 involved in catalysis: chain initiation and elongation • b' 1407 binds to the DNA template • 613 directs the enzyme to the promoter • 91 required to restore denatured RNA polymerase in vitro to its fully functional form

34. 4) Post-transcriptional modification: The newly synthesized precursors of rRNA and tRNA in bacteria undergo a series of process. A) Processing of rRNA: the 16S, 23S, and 5S rRNAs in prokaryotes are produced by cleavage of a rRNA precursor, catalyzed by ribonuclease III. Additional processes include methylation of bases and sugar moieties of some nucleotides.

35. Processing of rRNAs

36. B) Processing of tRNA: • The removal of the 5’ end of tRNA precursors is catalyzed by RNase P. RNase P is a ribozyme consisting of RNA that possesses enzyme activity. • Other processes include the addition of nucleotides (CCA) to the 3’-end of tRNA, and formation of some unusual residues such as pseudo-U, I, T, methyl-G, and DHU, etc.

37. Modification of some residues in tRNAs

38. 4) Inhibition of transcription: • Rifampicin: an antibiotic that specifically inhibits the initiation of transcription by blocking the formation of the first several phosphodiester bonds in RNA biosynthesis. • Streptolydigin: binds to bacterial RNA polymerase and inhibits elongation of RNA chain. • Actinomycin D: binds to DNA and prevents transcription (at low concentrations it doesn't affect DNA replication)

39. 2. RNA biosynthesis in eukaryotes • RNA polymerases in eukaryotes: three enzymes, each of which contains 12 or more subunits. Polymerase location RNAs transcribed Pol I nucleolus 28S, 18S, 5.8S rRNA Pol II nucleoplasm pre-mRNA, snRNA Pol III nucleoplasm tRNA, 5S rRNA, U6 snRNA, 7S RNA

40. 2) Process of eukaryotic RNA synthesis A) Initiation: similar to Pribnow box, a start site consensus (called TATA box) at –25 is required for the recognition by RNA polymerase in eukaryotes.

41. Pol II requires several transcription factors to start transcription: TFII-A: to stabilize the TFIID-TATA box complex; TFII-B: to link Pol II to the initiation complex; TFII-D: to recognize and bind to the TATA box; TFII-E: to interact with Pol II and TFII-B; TFII-F: to form Pol II-TFIIF complex. It also has DNA helicase activity; TFII-H, -J: to form the initiation complex.

43. B) Elongation : after the initiation complex has formed, the RNA polymerase catalyzes transcription in a 5’3’direction, using the (-) DNA strand as template. • Soon after the 5’end of the extending RNA chain appears from the polymerase complex, a cap structure is added at the end.

44. Cap structure of mRNA 7-methylguanylate

45. C) Termination: Two mechanisms may cause termination of RNA transcription: • A hairpin structure formed at the 3’end of the nascent RNA causes stop of transcription, as is seen in the prokaryotic RNA synthesis. • A stop signal sequence, AAUAAA, near the 3’end results in the recognition and binding by a specific endonuclease, which cleaves the nascent RNA chain and stops transcription. The newly synthesized mRNA precursor is then added a poly A tail by poly A polymerase.

46. Cleavage and polyadenylation of a mRNA precursor

47. 3) Processing of eukaryotic RNA precursors: • Gene organization: protein-coding genes in eukaryotic DNA are organized in a discontinuous fashion. The protein-coding sections are called “exons”, which are interrupted by noncoding sections called “introns”.

48. B) RNA splicing: a process in which introns of a pre-mRNA are removed to produce a functional mRNA.

49. C) Steps in RNA splicing: usually the exon-intron boundaries are marked by specific sequences. The intron starts with GU and ends with AG. Intron

50. Formation of a lariat intermediate: the phosphodiester bond of the 5’ splice site is attacked by the 2’-OH of the residue A in the branch point, forming a 2’5’bond and releasing the exon 1 with a new 3’-OH end. • Connection of exons: The new 3’-OH end attacks the phosphodiester bond at the 3’splice site causing the two exons to join and releasing the intron.