1. Lecture 6 of Introduction to Molecular Biology 生理所 蔡少正
Transcription
Lecture 6 of
Introduction to Molecular Biology
生理所 蔡少正

2. Enzymatic Synthesis of RNA
Precursors in RNA synthesis: rNTPs
Free 3’OH react with 5’ phosphate of rNTPs
Complimentary to DNA minus strand
RNA chain grows from 5’ to 3’ direction
Catalyzed by RNA polymerase
Four steps: binding of RNA polymersae to promoter, initiation, chain elongation, and termination

3. RNA Polymerases
--- Common: complex structure containing multiple subunits (two large subunits [>100kD] and several small subunits); do not need primer for initiation
--- Differences:

4. Promoter(I)
A region of DNA involved in binding of RNA polymerase to initiate transcription.
TATA box: (TATAA/TAA/T)
25 bp upstream of transcription start site. locating transcription initiation site, maintain promoter activity
CATA box
TATA less promoter: GC box (house-keeping genes; developmentally regulated genes)

5. Promoter(II) Upstream element: Initiator: (PyPyANT/APyPy)
(1) GC box (CCGCCC): usually multiple copies;
orientation independent.
(2) Cat box (CCAAT): CCAAT-binding transcription factor (CTF)
CCAAT/enhancer binding protein (C/EBP)
Initiator: (PyPyANT/APyPy)
conserved sequences around the transcription start site;
required for optional transcription activity
Downstream element:

6. General Transcription Factors
Definition: combine with RNA polymerase to form a preinitiation
complex that is competent to initiate transcription as soon as
nucleotides are available
Class II Factors
TFIIA, TFIIB, TFIID,
TFIIE, TFIIF, TFIIH
TBP (TATA
box binding protein)

7. Enhancer
A cis-acting sequence that increases the utilization of eukaryotic promoters; can function in either orientation
and in any location (upstream or downstream) relative to the promoter.

8. Termination:
(a) RNA polymerase I: Termination occurs at a discrete site > 1000 bp downstream of the mature 3' end, which is generated by cleavage. Termination involves recognition of an 18 bp terminator sequence by an ancillary factor.
(b) RNA polymerase III: Termination usually occurs at a U run (U4 or U5) within a G-C rich region.
(c) RNA polymerase II: Termination involves a consensus sequence AAUAAA for cleavage to generate a 3' OH end for polyadenylation and some small RNAs (e.g. U7 sn RNA).

9. Post-transcriptional Modification (RNA processing):
All the RNA molecules are modified after transcription. Both tRNA and rRNA molecules are excised from large primary transcripts.
Transcription

10. Capping
The 5' terminus of a mRNA molecule carries a methylated guanosine derivative, 7-methylguanosine (7-MeG), in an unusual 5'-5' linkage to the 5'-terminal nucleotide of the primary transcript, a structure called “Cap”.
Function of Capping
Increase mRNA stability
Enhance mRNA translation
Transport of RNA out of nucleus
Proper splicing of pre-mRNA

11. Polyadenylation
Most animal mRNA are terminated at the 3’ end with a poly(A) tract, which is added by poly(A) polymerase. The length of poly(A) segment can be from 50 to 250 nucleotides long.
Polyadenylation signal: AAUAAA
With poly(A)
No poly(A)

12. RNA Splicing
1. Most higher eukaryotic genes coding for mRNA and tRNA (few rRNA) are interrupted by unrelated regions called introns.
2. mRNA has 0-60 introns while tRNA has 0 or 1 intron
Transcription start site
Gene E1 I1 E2 I2 E3
hn RNA: Primary transcript
mRNA: Mature transcript

13. Splicing Signals The GU-AG rule: Get-U(you)-And-Go
Consensus Sequences in Mammals:
5’-AG/GUAAGU--intron--YNCURAC--YNAG/G3’

14. A Signal at the Branch Yeast: UACUAAC
Higher Eukaryotes: U47NC63U53R72A91C47

15. SnRNPs
Small nuclear RNA (snRNA): the agents that recognize the splicing signals
The SnRNAs are coupled with proteins in complex called snRNPs
U1 snRNA: Bind 5’ splice site of an mRNA precursor: necessary but not sufficient for splicing
U6 snRNP: Associated with the 5’ end of intron by base pairing; essential for splicing process
U5 snRNP brings together the last nucleotide in one exon and the first nucleotide of next exon
U4 snRNP base-pair with U6 until U6 is needed for splicing

16. Formation of Spliceosome

17. Alternative Splicing Occurs every 1 in 20 genes
Significance of alternative splicing:
--- Localization of protein: member-bound vs. secreted form proteins
--- Tissue specific expression
--- Different signaling pathway
--- Sex differentiation
--- Increase protein diversity

18. Self Splicing of Group I & II Introns
Group I intron can be removed without help from other protein
Group II intron self-splice using an A-branch lariat intermediate

19. RNA Editing
Trypanosomatid mitochondria encode incomplete mRNAs that must be edited before they can be translated
Editing occurs in the 3’ to 5’ direction by successive action of one or more guide RNAs