1. Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

2. DNA Transcription
Transcription produces an RNA chain identical in sequence with one strand of the DNA, sometimes called the coding strand.
This strand is made 5 ‘----3 ' and is complementary to (i.e., it base-pairs with) the template, which i s 3 ‘---5'.
The RNA like strand therefore is called the non template strand

3. Transcription Unit
A transcription unit is a sequence of DNA transcribed into a single RNA, starting at the promoter and ending at the terminator.

4. DNA Transcription Terminology
Promoter Seq: The sequence of DNA needed for RNA polymerase to bind to the template and accomplish the initiation reaction.
Start Point: Ist base where transcription start & then termination occur
Transcription Unit: Region b/w promoter to termination
Up Stream seq: Seq prior to the start point (-ve)
Down Stream Seq: Seq after the transcribe seq (+ve)
Primary Transcript: Immediate product of transcription

5. DNA Transcription Terminology
RNA synthesis in 5’ ’ direction
Replication Rate: 800bp/sec
Transcription Rate: 40bp/sec
Translation Rate: 15 AA/sec
RNA Pol. Create a transcription bubble where it binds to a promoter.

6. Start Point & Termination

7. Transcription Occurs by Base Pairing in a “Bubble" of Unpaired DNA
RNA polymerase separates the two strands of DNA in a transient "bubble" and uses one strand as a template to direct synthesis of a complementary sequence of RNA.
The bubble is 12 to 14 bp, and the RNA-DNA hybrid within the bubble is 8 to 9 bp.

8. Transcription Bubble
Transcription takes place in a bubble, in which RNA is synthesized by base pairing with one strand of DNA in the transiently unwound region.
As the bubble progresses, the DNA duplex re-forms behind it, displacing the RNA in the form of a single polynucleotide chain.

10. Transcription Bubble RNA Pol. Coding (sense) strand5’ 3’
Template (antisense) strand
Coding
(sense) strand
RNA
Pol.

11. Transcription Bubble RNA Pol. Coding (sense) strand5’ 3’
RNA
Pol. 3’ 5’
Template (antisense) strand

12. Transcription Bubble

13. Template recognition Initiation Elongation Termination
Transcription Stages
Template recognition
Initiation
Elongation
Termination

14. Transcription Stages
Template recognition: begins with the binding of RNA polymerase to the double-stranded DNA at a promoter to form a "closed complex". Then the strands of DNA are separated to form the "open complex“
Initiation: describes the synthesis of the first nucleotide bonds in RNA. The enzyme remains at the promoter while it synthesizes the first ~9 nucleotide bonds.

15. Transcription Stages
Elongation: Elongation involves the movement of the transcription bubble by a disruption of DNA structure, in which the template strand of the transiently unwound region is paired with the nascent RNA at the growing point.
Termination: Involves recognition of the point at which no further bases should be added to the chain. To terminate transcription.

16. Transcription Stages
The enzyme binds to the promoter and melts DNA and remains stationary during initiation; moves along the template during elongation; and dissociates at termination.

17. Transcription Stages

19. Bacterial RNA Polymerase Consists of Multiple Subunits
Bacterial RNA core polymerases are 400 kD multi subunit complexes with the general structure
Catalysis derives from 13' subunits.
About 13,000 RNA polymerase molecules are present in an E. coli cell, although the precise number varies with the growth conditions

20. Bact RNA Polymerase 2,000-5,000 are synthesizing RNA at any one time
The complete enzyme or holoenzyme in E.Coli has a M.Wt. ~465 kDa

21. Bacterial RNA Polymerase Sub units

22. Bacterial RNA Polymerase Sub units

23. Bacterial RNA Polymerase Sub units

24. RNA Polymerase Core & Sigma Factors
The Holoenzyme can be divided into 2 components
Core Enzyme: The core enzyme has four polypeptide subunits: alpha (a), beta (b), beta' (b'), and omega (w) in the stoichiometry a2bb'w
Core enzyme has the ability to synthesis RNA but can’t initiate transcription
It can’t distinguished b/w promoter & other DNA seq

25. RNA Polymerase Core & Sigma Factors
Sigma Factor: It directs RNA polymerase to the promoter and ensures that transcription is initiated only where it is supposed to be initiated.
Sigma factor release when chain reaches bases

26. Core & Sigma Factors

27. RNA Polymerase Core & Sigma Factors

28. RNA Pol Conformational Changes
RNA Pol initially binds to DNA it covers bases (starting -55 to +20)
Then shape of RNA Pol changes from initiation to elongation, which cover ~ 60 bases

29. RNA Pol Conformational Changes

30. RNA Pol Function

31. Thanks