1. Hebrews 1:1-2
1 God, who at sundry times and in divers manners spake in time past unto the fathers by the prophets,
2 Hath in these last days spoken unto us by his Son, whom he hath appointed heir of all things, by whom also he made the worlds;

2. Transcription From DNA To RNA
Timothy G. Standish, Ph. D.

3. Introduction The Central Dogma of Molecular Biology
Cell
DNA
mRNA
Transcription
Polypeptide
(protein)
Translation
Ribosome

4. RNA Polymerase RNA Polymerase is a spectacular enzyme, functioning in:
Recognition of the promoter region
Melting of DNA (Helicase + Topisomerase)
RNA Priming (Primase)
RNA Polymerization
Recognition of terminator sequence

5. A “Simple” Gene Transcription Start Site 3’ Untranslated Region5’ 3’
Protein Coding Region
Promoter/
Control Region
RNA Transcript
Terminator
Sequence

6. Stages of Transcription
Initiation
Elongation
Termination

7. Prokaryotic Transcription Initiation
The a subunit of prokaryotic RNA polymerase is necessary for promoter recognition and binding of RNA polymerase to the promotor
Different a subunits allow recognition of different types of promoters thus the type of genes transcribed can be modulated by altering the types of a subunits which attach to RNA polymerase

8. Prokaryotic Transcription Initiation
RNA
Pol.  P1 P2
Different promoters
Heat Shock Gene
Constitutive Gene

9. Prokaryotic Transcription Initiation
RNA
Pol.  P1
Heat Shock Gene P2
Constitutive Gene
Different promoters

10. Eukaryotic Transcription Initiation
Proteins called transcription factors bind to the promoter region of a gene
If the appropriate transcription factors are present, RNA polymerase binds to form an initiation complex
RNA polymerase melts the DNA at the transcription start site
Polymerization of RNA begins

11. Initiation
T. F.
Promoter
RNA
Pol.
T. F.
RNA
Pol.
T. F. 5’
RNA

12. Initiation
T. F.
Promoter
RNA
Pol.
T. F.
T. F.
RNA
Pol. 5’
RNA

13. Transcription Termination
There are two types of termination:
Rho dependent requires a protein called Rho, that binds to and slides along the RNA transcript. The terminator sequence slows down the elongation complex, Rho catches up and knocks it off the DNA
Rho independent termination depends on both slowing down the elongation complex, and an AT-rich region that destabilizes the elongation complex

14. Termination Rho Independent
RNA
Pol. 5’
Terminator
RNA
Pol. 5’

15. Termination Rho Independent
RNA
Pol. 5’
RNA
Pol. 5’
Terminator

16. Termination Rho Dependent
Terminator
RNA
Pol. 5’ r
RNA
Pol. 5’ r
The terminator sequence slows RNA polymerase

17. Termination Rho Dependent
Terminator
RNA
Pol. 5’ r
Help, Rho
hit me!
RNA
Pol. 5’ r
Rho catches up with RNA polymerase

18. Termination Rho Dependent
Terminator
RNA
Pol. 5’ r r
RNA
Pol. 5’
The elongation complex disintegrates

19. Differences Between Transcription In Prokaryotes and Eukaryotes

20. Transcription And Translation In Prokaryotes3’ 5’ 5’
mRNA
RNA
Pol.
Ribosome
Ribosome

21. Eukaryotic Transcription
DNA
Cytoplasm
Nucleus
Nuclear
pores
RNA
Transcription G
AAAAAA
RNA
Processing
mRNA
Export G
AAAAAA

22. A “Simple” Eukaryotic Gene
Transcription
Start Site
5’ Untranslated Region
3’ Untranslated Region
Introns 3’ 5’
Exon 2
Exon 3
Int. 2
Exon 1
Int. 1
Promoter/
Control Region
Exons
Terminator
Sequence
RNA Transcript 3’ 5’
Exon 2
Exon 3
Exon 1
Int. 2
Int. 1

23. Processing Eukaryotic mRNA
3’ Untranslated Region
5’ Untranslated Region
Protein Coding Region 5’ G
5’ Cap 3’ 5’
Exon 2
Exon 3
Int. 2
Exon 1
Int. 1 3’
AAAAA
3’ Poly A Tail
Exon 2
Exon 3
Exon 1
Int. 2
Int. 1
RNA processing achieves three things:
Removal of introns
Addition of a 5’ cap
Addition of a 3’ tail
This signals the mRNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm

24. Common Splicing Mechanism
Intron
Exon 2
Exon 1 AG A GU 3’ 5’
U A C U A A C (Yeast)
18-40 BP
Branch site
Left (donor)
5’ splice site
Right (acceptor)
3’ splice site
Py80NPy80Py87Pu75APy95
(Animal-Subscripts indicate percent frequency)
The branch sequence allows identification of the 3’ splice site

25. Common Splicing Mechanism
Lariat Formation l
Yee ha!
Exon 2 A AG G U 3’ 5’
Exon 1
Intron
Lariat
Lariat

26. Common Splicing Mechanism
Exon 1
Exon 2 5’ 3’ A AG G U
Intron lariat
Following excision, the lariat is rapidly degraded

27. Common Splicing Mechanism
Exon 1
Exon 2 5’ 3’
Following excision, the lariat is rapidly degraded

28. The Spliceosome
Spliceosomes are structures that form within the nucleus to remove introns from eukaryotic hnRNA
This structure is large, on the order of a ribosome subunit
Like the ribosome, spliceosomes are composed of both protein and RNA

29. Wobble Base Pairing

30. The Rules of Codon Anticodon Base Pairing
Three things affect the way in which base pairing occurs between codons on mRNA and anticodons on tRNA:
How the two molecules “twist” when annealing - They are not free to form a perfect A helix
The environment of the Ribosome A site
Chemical modification of bases
These three factors change the usual base pairing seen in DNA and RNA, particularly at the first base of anticodons/third base of codons

31. Transfer RNA (tRNA) Anticodon3’ 5’
Acceptor Arm - A specific amino acid is attached to the 3’ end U* 9 26 22 23 Pu 16 12 Py 10 25
20:1 G*
17:1 A
20:2 17 13 20 G 50 51 65 64 63 62 52 C 59 y A* T 49 39 41 42 31 29 28
Pu* 43 1 27 U 35 38 36
Py* 34 40 30
47:1
47:15 46
47:16 45 44 47 73 70 71 66 67 68 69 3 2 7 6 5 4
TyC arm - y stands for pseudouridine
D Arm - Contains dihydrouridine
Extra Arm - May vary in size
Anticodon

32. Transfer RNA (tRNA) Dihydro-uridine O NH N Pseudo-uridine N O HN NH9 26 22 23 Pu 16 12 Py 10 25
20:1 G*
17:1 A
20:2 17 13 20 G 50 51 65 64 63 62 52 C 59 y A* T 49 39 41 42 31 29 28
Pu* 43 1 27 U 35 38 36
Py* 34 40 30
47:1
47:15 46
47:16 45 44 47 73 70 71 66 67 68 69 3 2 7 6 5 4
TyC arm - y stands for pseudouridine
D Arm - Contains dihydrouridine
Pseudo-uridine N O HN NH
Anticodon

33. Some Other Strange tRNA Bases

34. Base Pairing Guanine And CytosineH O N
Guanine - N O H
Cytosine + + - - +

35. Base Pairing Adenine And UracilH - +
Adenine N O H + -
Uracil

36. Base Pairing Adenine And CytosineH
Cytosine - + N H - +
Adenine

37. Base Pairing Guanine And UracilH O N
Guanine + - N O H + -
Uracil

38. Wobble Base Pairing Guanine And UracilH
Uracil + - H O N
Guanine + -

39. Base Pairing Adenine And 2-Thiouracil+
Adenine N S O H + -
2 Thio-
uracil

40. Wobble Base Pairing Guanine And 2-Thiouracil+ - S N O H
2 Thio-
uracil +
2-Thiouracil forms only one hydrogen bond with guanine which is not enough to form a stable pair in the environment of the ribosome A site

41. Wobble Base Pairing Inosine And CytosineH
Inosine + - N O H
Cytosine - +

42. Wobble Base Pairing Inosine And UracilH
Uracil + - O N H
Inosine - +

43. Wobble Base Pairing Inosine And AdenineH
Inosine + - N H - +
Adenine

44. The Wacky Rules of Wobble Base Pairing
First anticodon base:
Third codon base:
A or G
----- A G U
C or U
C U or G U
2-S-U C A G I

45. Wobbling and tRNA Numbers
The net effect of wobble base pairing is to reduce the number of tRNAs that must be produced by a cell
In reality cells do not make 61 different tRNAs, one for each codon
Many tRNAs have anticodons that anneal to several different codons\
Codons are known for which there are more than one tRNA, although each tRNA carries the same amino acid (i.e., methionine)

46. The
End