1. Transcription in prokaryotes

2. [1]
In prokaryotes, only one RNA polymerase is responsible for the synthesis of all RNA (except for the RNA primers required for replication)
Compare the mechanism of action of RNA polymerase and DNA polymerase.

3. s a a b’ b  RNA POLYMERASE Core Enzyme 2a, b, b’ and  Holoenzyme
2a, b, b’ and  and s b’ b 

4. Similarities
Substrate for both - nucleoside triphosphates
Pyrophosphate released for every NTP added
Always in the 5’3’ direction
Differences
Recognizes the site for initiation of transcription
Ribose sugar and uracil
No primer required
No proofreading activity

5. [2]
For genes to be transcribed by RNA polymerase the appropriate initiation site must be recognized.
How does the RNA polymerase know where to start transcription?
How does the sequence of the Pribnow box relate to its function?

6. Transcription start site
PROMOTERS
-35 sequence
Pribnow box
TTGACA
TATAAT
box 5’ 3’ 3’ 5’ +1
~19 bases
~7 bases
Transcription start site

7. [3]
Each of the two DNA strands can serve as templates for transcription
What determines which of the two DNA strands will be transcribed??
Can both the DNA strands be transcribed??
Which is the sense (coding) and anti- sense (noncoding) strand ??
The RNA has the same sequence as which one of the two DNA strands?

8. [3] What determines which of the two DNA strands will be transcribed??
the location of the promoter
Can both the DNA strands be transcribed??
Yes, but for a given gene only one strand will be the template
Which is the sense (coding) and anti- sense (noncoding) strand ??
Sense strand = the same sequence as the mRNA, contains the promoter
antisense = acts as the template, complementary sequence to the mRNA.

9. Transcription

10. [4] INITIATION, ELONGATION, TERMINATION
Three separate stages of transcription
INITIATION,
ELONGATION,
TERMINATION

13. TERMINATION r - independent

14. TERMINATION r - independent

15. TERMINATION r - independent

16. Action Of Antibiotics on Transcription
Rifampin - binds to b-subunit of prokaryotic RNA polymerase
tuberculosis
Dactinomycin – binds to DNA, prevents the movement of RNA polymerase
Tumor chemotherapy

17. TRANSCRIPTION -EUKARYOTES

18. RNA Polymerases in Eukaryotes
RNA POLYMERASE I - rRNA
RNA POLYMERASE II - mRNA, snRNAs
RNA POLYMERASE III - tRNA and other small RNAs

19. Eukaryotic gene structure
= intron (untranslated)
= exon (translated)
Pre-mRNA 5’ 3’
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 1
Exon 4
Exon 3
Exon 2
Exon 5
mRNA
PROTEIN

20. RNA Polymerase II 10-12 subunits
The largest subunit contains a C-terminal region or domain (CTD) with 52 repeats of the amino acid sequence PTSPSYS
T = threonine
S = serine
Y = tyrosine
Can be phosphorylated

21. RNA PLOYMERSE II PROMOTERS
Transcription start site
CAAT
box
TATA
box 5’ 3’ 3’ 5’
-70
-30
-20 +1
45-55 bases
25 bases

22. Transcription factors
Any protein that regulates transcription but is not a part of RNA polymerase, is a transcription factor
Six general transcription factors (GTFs) required for all promoters
TFIIA
TFIIB
TFIID
TFIIE
TFIIF
TFIIH
+ RNA Polymerase II = Preinitiation complex

23. PREINITIATION COMPLEX (PIC)
Pol II F E H
TFIID B A
TATA box
PREINITIATION COMPLEX (PIC) A
TFIID B F
TFIIB binds to TFIID and acts as bridge between Pol II and TFIID
Complex of several proteins, TATA-Binding Protein (TBP) and ~12 TBP Associated Factors (TAFs)
TFIIF binds Pol II and targets its to the promoter
Recruits TFIIH
TFIIA stabilizes the DNA and TFIID interaction E H
Promoter melting, kinase activity

24. E H
TFIID B
Pol II A F

25. GTFs only produce a basal level of transcriptionH
TFIID B
Pol II A F
mRNA
GTFs only produce a basal level of transcription

26. Enhancers/Silencers Upstream or downstream
Close to the promoter or thousands of base pairs away
On either of the two strands of DNA
Act through intermediary or gene specific transcription factors proteins
Enhancers activate transcription
Silencers deactivate transcription

27. Enhancers/Silencers

28. Response Elements
Enhancers contain response elements that are responsive to certain metabolic factors
Cyclic AMP response element (CRE)
Glucocorticoid response element (GRE)
Heat shock element (HSE)
REs bind transcription factors produced under certain cell conditions to activate several related genes

29. a-amanitin and RNA Pol II

30. Post Transcriptional Modification – mRNAs 5’ Capping

31. Post Transcriptional Modification – mRNAs Poly-A tail

32. Post Transcriptional Modification – mRNAs SPLICING

34. Post Transcriptional Modification - rRNAs
RNA Polymerase I

35. Post Transcriptional Modification - tRNAs

36. THE END!