1. Genetic information transfer------ (RNA transcription-转录)
RNA biosynthesis
Lecturer: Jiao Li
Phone:

2. Objectives Understand RNA polymerase involved in transcription.
After completion of this section, you should be able to :
Understand RNA polymerase involved in transcription.
Describe the process of RNA transcription and post-
transcriptional modifications.
4. Appreciate the types of introns and mechanism of splicing.

3. reverse transcription
Central dogma
replication
transcription
translation
RNA
Protein
DNA
RNA replication
reverse transcription

4. Outline of the section 1. Transcription system
2. Biosynthesis process of RNA
3. Post-transcriptional modifications

5. RNA DNA RNA transcription
A reaction in which a template strand of DNA is utilized by specific RNA polymerase to generate one kind of RNA.
Getting the message out!
DNA
transcription
RNA

6. Types of RNAs RNA type size function Coding RNA:
mRNA variable carrying information of protein sequence
Non-coding RNA:
Transfer-tRNA small carrying amino acid to growing polypeptide
Ribosomal-rRNA variable major and active component of ribosome,
most abundant RNA
Small nuclear-snRNA small RNA processing
Small nucleolar-snoRNA small RNA processing
Small cytoplasm-scRNA small SL-RNA- component of SRP
Micro-RNA nt gene regulation
Small interference-siRNA nt gene regulation

7. Electrophoresis of total RNA from eukaryotes

8. Section1 Transcription system

9. Transcription system Substrates: NTP (ATP, UTP, GTP, CTP)
Template: DNA
Enzyme: RNA polymerase, RNA-pol
Other protein factors

10. §1.1 RNA polymerase- RNA pol
a. RNA pol in prokaryote
In prokaryotic (bacteria) all types of RNA are synthesized by only one species of RNA polymerase.

11. Transcription specificity
Subunits of RNA polymerase in prokaryote
subunit gene MW number function
α rpo A Binding upper promoter element
and regulatory factors
β rpo B Polymerase activity
β′ rpo C Binding to DNA(nonspecifically)
σ rpo D Binding specific promoter regions
ω Not known    
Core enzyme
Holoenzyme
Synthesize RNA
Transcription specificity
Promoter recognization
σ70 : common promoter
σ32 : HSP promoter

12. Rifampicin inhibits DNA-dependent RNA polymerase in bacterial cells
Mechanism:
by binding its β-subunit of RNA polymerase
Bactericidal antibiotic
Separate
Rifampicin sensitive
Rebuild
Rifampicin resistance
Rifampicin resistance

13. §1.1 RNA polymerase- RNA pol
b. RNA pol in eukaryote
“death cap” mushroom
CTD: carboxyl terminal domain -, c-terminus of the largest subunit of RNA polⅡ , which consists of repeat sequence-Tyr Ser Pro Thr Ser Pro Ser.
Tyr ,Ser and Thr residues in CTD can be phorsphorylated, which is involved in initiation of transcription.
RNA polⅡ

14. Characteristics of RNA pol
No primer
No exonuclease activity-no proofreading function

15. §1.2 DNA template
Structural gene : codes for any RNA or protein product.
template strand: antisense strand. Template for guiding RNA synthesis.
coding strand: sense strand. Strand complementary to template strand, same with RNA product. 5 3
template strand
coding strand
Structural gene
Transcription direction

16. 5′···GCAGTACATGTC ···3′
Coding strand
3′··· c g t g a t g t a c a g ···5′
Template strand
Transcription
mRNA
5′···GCAGUACAUGUC ···3′
Translation
N······Ala · Val · His · Val ······C
Protein
mRNA: sequence is the same to coding strand.

17. Asymmetric transcription
Asymmetric transcription: for one structural gene, only one DNA strand serves as template for RNA synthesis.
Both DNA strands can serve as template.

18. Specific sequences on DNA are important for transcription
RNA pol protection assay
RNA pol Ⅱ
Digested by nuclease
Release of binding fragment
It shows RNA pol binding sequence is important for transcription ---- promoter

19. Important sequences for transcription: Promoter Enhancer terminator
Structural gene
Regulatory sequence
Promoter
Structrual gene
Terminator
Transcrition unit
Operon

20. Promoter
Promoters：locate near the genes they regulate, on the same strand and typically upstream of the antisense strand.
Direct the level and accuracy of transcription of a given gene.

21. Promoter in prokaryote
Initiation site
T T G A C A
A A C T G T
-35 区
RNA pol binding site
TATA box
(Pribnow box)
T A T A A T Pu A T A T T A Py
-10 区 1
-30
-50 10
-10
-40
-20
5 
3 
(RNA-pol
recognition site) 5
RNA pol binding region
Structral gene 3
Promoter in pro：
RNA pol 识别和结合的部位
-35: recognition site
-10: binding site
+1: initiation site

22. Promoter in eukaryote
TATA box
CAAT box
GC box
enhancer
Cis-acting element in eukaryote-promoter：it is where RNA pol binds by TF.
structural gene
-GCGC---CAAT---TATA
Initiation site
Cis-acting element： specific DNA sequence which can regulate gene expression.
Transcription factors –TF：protein which can bind to DNA sequence to control gene transcription.

23. TF types recognized by RNA polⅡ
① General transcription factors : commonly present and interact with promoter region of genes transcribed by RNA polⅡ---TF Ⅱ A, TA Ⅱ B , TF Ⅱ D ,TF Ⅱ E , TF Ⅱ F and TF Ⅱ H .
② Upstream transcription factor: recognizing and binding specific sequence of gene surrounding promoter region.
③ Inducible factor: can stimulate or repress gene transcription.

24. b. Terminator
Terminator: locates downstream of structural gene and stops transcription.
Terminator in prokaryote: form stem-loop structure .
Terminator in eukaryote: no
真核生物 mRNA 基因无终止子。

25. c. Enhancer

26. Section2 Biosynthesis process of RNA

27. Initiation Elongation Termination §2. RNA transcription process
Sequential actions
Initiation
Elongation
Termination

28. Initiation Recognize the starting point Separate dsDNA
§2. 1 Transcription of prokaryotes
Initiation
Recognize the starting point
Separate dsDNA
3. Initiation complex

29. Initiation 1. RNA-pol holoenzyme(2) binds template
2. DNA unwinding
1. RNA-pol holoenzyme(2) binds template
3. Forming initiation complex
4. Forming the first phosphodiester bond
Transcription initiation complex
G or A
RNApol (2) - DNA - pppGpN- OH 3
5-pppG -OH + NTP  5-pppGpN - OH 3 + ppi

30. Elongation (NMP) n + NTP  (NMP) n+1 + PPi
5. Once the first 10 nucleotides are incorporated,  factor dissociates from holoenzyme and the core enzyme left keeps proceeding to extend RNA.
factor dissociate
6. RNA product is elongated under catalysis of core enzyme.
(NMP) n + NTP  (NMP) n PPi

31. Transcription bubble：
Positive supercoil
Negative supercoil
RNA pol
Direction of transcription
Transcription bubble：
RNA-pol （core enzyme） ···· DNA ···· RNA

32. RNA DNA RNA polymerase ribosome
Electron micrograph of rRNA transcription
“leather” formed during RNA transcription. 5 3
DNA
ribosome
RNA
RNA polymerase
Leather shaft

33. Termination
Transcription is terminated by signals within the RNA sequence
In bacteria, terminators come in two types:
Rho-dependent termination
Rho-independent termination.

34. Rho-dependent ATP ρfactor：protein, 275 kD .
RNA pol stop at termination point(100nt away from ρbinding site )
ρfactor：protein, 275 kD .
ρ factor can bind ‘C’ rich region in newly synthesized RNA.
ρ factor has ATPase and RNA-DNA helicase activity---release RNA from RNA-DNA hybrid double chains.

35. Rho-independent
Stem-loop structure in RNA stops RNA pol from elongating.
Poly A-U in RNA relase RNA from RNA-DNA.
Stem-loop structure
A-U

36. RNA Synthesis

37. §2. 2 Transcription of eukaryotes
Differences of transcription between prokaryotes and eukaryotes
① RNA pol in eukaryotes:Ⅰ ,Ⅱ, Ⅲ
② RNA pol Ⅱis directed to promoter by other transcription factors.
③ There are more multiple cis-acting elements in upstream of starting site and more proteins involved in initiation.
④ Termination is coupled with modification.

38. Initiation
Transcription factor II（TFII）: TFs which direct RNA pol II to promoter and help to initiate transcription.
Factor Subunit Function
TFIID TBP, TAFs Identifing and binding TATA box
TFIIA Stabilizing binding of TFIID
TFIIB Forming ternary complex with A and D
TFIIF Binding with RNA pol II
TFIIE Recruiting TFIIH
TFIIH Helicase, kinase activity
TBP: TATA box binding protein, one subunit of TFIID
TAFs: TBP associated proteins.

39. TFII D binds TATA box
TFIIA and TFII B join to form ternary complex
TFII F binds RNA pol II and join the complex
The addition of TFII E and TFII F is the final step
TFII H unwind DNA strands and phosphates CTD
RNA pol II and TFIIs form pre-initiation complex (PIC),PIC is similar to RNA pol holoenzyme in prokaryote.
CTD

40. In vivo, Transcription Initiation Requires Additional Proteins
Assembly of the pre-initiation complex in presence of mediator, nucleosome modifiers and remodelers, and transcriptional activators.

41. Elongation nucleosome Direction of transcription RNA-Pol RNA-Pol
Dissemble of nucleosome
Dissociation of nucleosome
Reassembly of nucleosome
RNA-Pol
Reassemble of nucleosome
RNA-Pol

42. Termination —— Coupling with the addition of poly A tail. 3tail
5 AAUAAA--
AAAAAAA······ 3 mRNA
3tail
5------AAUAAA-
Nuclease
-GUGUGUG
RNA-pol 5 3 5
AATAAA GTGTGTG 3
Site for polyadenylation

43. Section 3 Posttranscriptional processing

44. Posttranscriptional processing
After transcription, the formed immature RNA will undergo modifications to be mature and functional.
Processing types:
① Cleavage and Splicing
② Terminal addition
③ Modification
④ RNA editing

45. §3. 1 posttranscriptional processing in prokaryote
rRNA
methylation
Methyl group
cleavage

46. §3. 1 posttranscriptional processing in prokaryote
tRNA
cleave
addition
modification
Cleavage
Terminal addition
modification

47. §3. 2 posttranscriptional processing in eukaryote
rRNA 转录 剪接
18S - rRNA
5.8S和28S-rRNA
rDNA
Intron
28S
5.8S
18S
45S - rRNA

48. tRNA Modifications （1）Methylation example：A  Am （2）Reductive reaction
Exo and endonuclease
Modifications
example：A  Am
（1）Methylation
（2）Reductive reaction
example：U  DHU
tRNA nt transferase
（3）Translocation reaction
example：U  ψ
modifications
（4）Deamination
example：A  I

49. Types of processing : mRNA ① Capping ② Addition of poly A tail
③ Splicing
④ Methylation
⑤ RNA editing

50. ① Capping
The addition of 5cap (m7GpppGp —)
5’ to 5’ connection

51. 5 pppGp… 5 ppGp… 3 GpppGp… 3 m7GpppGp… Pi ppi
The addition of 5cap
5 pppGp…
3 GpppGp…
pppG
ppi
Guanosine transferase
3 m7GpppGp…
Methyl group transferase
SAM
5 ppGp…
phosphatase Pi

52. 2. Permit initiation of translation (specific, where translation
Cap O
Cap Ⅰ
Cap Ⅱ
Function:
1. Help to stabilize mRNA
2. Permit initiation of translation (specific, where translation
should begin).

53. ② Addition of poly A tail
Specific endonuclease cuts at modification site.
PolyA polymerase catalyzes without template- untemplated extension.
polyA :∽more than 250 nt .
Histone: no poly A tail.
Polyadenylate polymerase

54. Roberts and Sharps found interrupted genein 1977 separately.
R looping
Mature mRNA hybrids with DNA

55. ③ Splicing C A B D Coding region: A, B, Cand D Non-coding area
Split gene
A eukaryotic gene in which the coding sequence is divided into two or more exons that are interrupted by a number of noncoding intervening sequences (introns). Also known as interrupted gene. C A B D
Non-coding area
Coding region: A, B, Cand D

56. Exon and Intron
Exon
DNA sequence present in final mature RNA product and codes for primary sequence of protein.
Intron
DNA sequence not present in final mature RNA product and is removed during splicing process.
hnRNA
Transcription, capping and tail
Splicing
mRNA

57. —— Introns are removed and exons are joined.
Splicing of mRNA
—— Introns are removed and exons are joined.
Exon upstream-AGGUAAGU A (Py)nNCAGG-exon downstream
5’Splicing site
3’ Splicing site
Branch site
intron
snRNA（U1,2,4,5,6）+ protein = snRNP ( small nuclear ribonucleoprotein)
snRNP+hnRNA= Splicesome
Exon
Exon
Intron

58. Two steps of transesterification reactions
Nucleophilic attack
5’-2’ bond
Mechanism of action of mRNA splicing

59. Transcription and posttranscription of egg albumin
Egg-albumin gene
Transcription
hnRNA
Modification in 5’and 3’
hnRNA splicing
Muture mRNA
Transcription and posttranscription of egg albumin

60. Alternative splicing Cross-exon splicing
U1bind to 5’ end of downstream intron
Cross-exon splicing
U2 bind to branch site A

61. Alternative splicing of tropomyosin (TM)
One gene multiple mRNAs multiple proteins
（Isoforms）

62. Faulty splicing can cause disease
β: There is a point mutation in exon-intron junction of β-hemoglobin chain, leading to abnormal splicing of intronand further diminished or absent synthesis of β-chain protein of hemoglobin.

63. （truncated protein, mw: 240,000） mRNA editing
④ Methylation
⑤ RNA editing
Apo B gene in human
mRNA（14500 nt）
Liver: apo B100
（full protein, mw: 500,000）
Intestine: apo B48
（truncated protein, mw: 240,000）
mRNA editing
RNA editing: a kind of process which changes sequence of RNA transcript by untemplated insertion, deletion or substitution. It can alter amino acid sequence of protein coded which differs from that predicted by DNA sequence.
• Differential RNA processing

64. Structure of mature mRNA
UTR: untranslated region

65. Evolution or Destined? Function of introns
1. Can be further processed after splicing to generate noncoding RNA molecules.
2. Alternative splicing is widely used to generate multiple proteins from a single gene.
3. Represent mobile genetic elements and may be regarded as examples of selfish DNA.
Provide more possibilities for genome!

66. Section 4 RNA replication

67. RNA replication （RNA 复制）
RNA virus: Viruses need an RNA-dependent RNA-polymerase (RDRP) to replicate their RNA, but animal cells do not seem to possess a suitable enzyme. ——RNA 复制酶为RNA病毒复制所需
RDRP: no primer and no proofreading function.
Plus-stranded RNA viruses（正单链RNA病毒 including retrovirus）
poliovirus (脊髓灰质炎病毒) 
SARS病毒
flaviviruses （虫媒病毒）
Negative-stranded RNA viruses（负单链RNA病毒）
influenza virus (流感病毒)
measles virus, mumps virus, (麻疹，腮腺炎病毒)
rabies virus (狂犬病毒)
Double-stranded RNA viruses（双链RNA病毒）
rotaviruses (轮状病毒-呼肠孤病毒reovirus)
'reɪbiːz; -ɪz

68. Replication of RNA virus
Plus-stranded RNA viruses ： the same as mRNA and functions as mRNA .
Negative-stranded RNA viruses ： complementary to mRNA and need to be copied to plus-sense mRNA.
Double-stranded RNA viruses ： cannot function as mRNA and need RDRP.

69. RNA synthesis in viruses
Retrovirus-逆转录病毒
RNA synthesis in viruses

70. Summary
There are 1 major enzyme involved in RNA transcription in prokaryote; while there are 3 major enzymes involved in RNA transcription in eukaryote.
The process of RNA transcription: initiation; elongation; termination
Types of RNA modification: 5’; 3’; splicing; RNA editing; base modification.