1. RNA Synthesis (Transcription)
Dr. Shumaila Asim Lecture # 2

2. RNA Synthesis (Transcription)
Introduction
First step in gene expression
RNA is involved in the transfer of genetic information stored in DNA to proteins
Process of DNA directed RNA synthesis (copying process) (genes in DNA make RNA copies)
Synthesis of an RNA strand with a base sequence complementary to one strand of DNA (template) and identical in sequence to the other strand

3. RNA Synthesis (Transcription)
Selective process; one particular gene or groups of genes are transcribed
Specific sequences indicate the beginning and end of DNA segment to be transcribed
Some proteins are also involved in this “selectivity of the process”

4. RNA Synthesis (Transcription)
Occurs in 5 − 3 direction
Catalyzed by RNA polymerase I, II & III (DNA dependent RNA polymerases (RNAPs)
Each RNAP consists of two large subunits and many small subunits

5. Numbering
DNA A – T – A – C – G – T – T – G – A – C – G -----
RNA A – A – C – U – G – C -----
Position +1 of a gene is the base that is complementary to the first base of the 5 end of the RNA transcript of that gene (initiation point)
Sequences preceding first base are numbered negatively and are said to be upstream of the initiation point
Sequences following the first base are numbered positively – downstream

6. Specific Sequences
Promoters – Specific areas or sequences of DNA for initiation (different for different RNAPs) ,these are
Pribnow box (TATA box) (Prokaryotes)
Goldberg-Hogness box or TATA box or consensus sequence (Eukaryotes)

7. Specific Sequences Terminators – Sequences for termination
Enhansers & silencers – Sequences for increasing or decreasing rate of transcription
Transcription Unit – Region between promoter and terminator (includes sequences for initiation, elongation & termination)

8. Requirements Template = DNA single strand
Substrates = Ribonucleotides (ATP, GTP, UTP, CTP)
Initiation factors
Transcription factors [Tf – II or GTF (General Transcription factors)] (Tf-II include Tf-IIA, Tf-IIB, Tf-IIC, Tf-IID, Tf-IIE, Tf-IIF, Tf-IIH)
Tf-IID contains TATA binding protein (TBP) and 14 TAFs (Transcription activating factors)
Activators, Co-activators and components of PIC (pre-initiation complex)

9. Requirements
Enzymes = DNA-dependent RNA polymerases [RNA polymerase (RNAP) I, II, III]
RNAP–I synthesizes rRNA
RNAP–II synthesizes mRNA
RNAP–III synthesizes tRNA, 5S rRNA
Each RNAP holoenzyme consists of Core-enzyme + one or more sigma ()factors
Sigma () factors assist the core enzyme to recognize promoter region

10. Requirements
RNAP is zinc containing metallo-enzyme, requires Mg++ ions for its activity
Core enzyme consists of two identical -subunits, one each ,  and  subunits
-subunit is thought to be the catalytic subunit (the growing chain of RNA remains attached to this subunit)

11. Generalized Functions of RNAPs
Unwinding the DNA in front,
Synthesis of RNA,
Re-winding it behind,
Dissociating the growing RNA chain from the template and
Perform proofreading.

12. RNA Synthesis (Transcription)
A cyclic process, involving RNA chain initiation, elongation & termination
RNA polymerase attaches to and initiates transcription at a distinct site, the promoter (a special DNA sequence)
GTFs (Tf-II) facilitate promoter-specific binding of RNAPs to form pre-initiation complex (PIC)
Other proteins (co-activator proteins) regulate the rate of transcription initiation

13. RNA Synthesis (Transcription)

14. RNA Synthesis (Transcription Steps)
Initiation:
Formation of transcription bubble
Protomer–Polymerase complex (Pre-initiation complex)
PIC formation leads to Structural changes (melting or unwinding of ssDNA) producing “bubble” known as “transcription bubble”

16. RNA Synthesis (Transcription Steps)
First nucleotide (always purine ATP or GTP) attaches to enzyme (RNAP) through its 5-end
RNAP now catalyzes the formation of a phospho-diester bond between the first nucleotide (free 3-end) and the 5-position of next entering nucleotide

17. RNA Synthesis (Transcription Steps)
Synthesis of RNA continues in 5 − 3 direction in a sequence dictated by DNA template (base pairing rule) anti-parallel to DNA template
Pyrophosphate (liberated in each addition) is rapidly degraded to two inorganic phosphates making the RNA synthesis irreversible

18. RNA Synthesis (Transcription Steps)
Elongation:
After addition of 10 – 20 ribonucleotides, RNAP undergoes a second structural change
Moves away from the “promoter”
Progresses along DNA molecule – thereby producing “bubble” (DNA unwinding) along DNA template as RNA synthesis proceeds (elongation)

19. RNA Synthesis (Transcription Steps)
Once the RNA polymerase has synthesized a short stretch of RNA (approximately ten bases), it shifts into the elongation phase.
This phase requires further conformational change in RNA polymerase that leads it to grip the template more firmly.

20. RNA Synthesis (Transcription Steps)
Termination:
Specific sequences or specific signals are recognized by Rho factor ( factor) of RNAP (in Prokaryotes)
Rho factor attaches to DNA; the RNAP cannot move further,
Dissociates from DNA and
Newly synthesized RNA is released.

21. RNA Synthesis (Transcription Steps)
In eukaryotes, the termination is independent of Rho factor
Completed RNA chain and RNAP are released from template
RNAP holoenzyme reforms, finds another promoter and synthesis of new RNA starts

23. Modifications (RNA) in primary transcription
Terminal additions,
Base modifications,
Trimming,
Internal segment removal and splicing  lead to    functional RNA molecule

24. Modifications (RNA) in primary transcription
RNA polymerase I synthesizes large ribosomal RNAs in nucleolus
RNAP–II synthesizes the precursors of mRNAs
RNAP–III synthesizes small RNAs like tRNAs, the small 5S rRNA

25. Post-transcriptional RNA Processing
A. rRNA Processing
45 S Precursor rRNA
5.8 S rRNA
18 S rRNA
28 S rRNA
Spacer sequences
specific
endonucleases
5 S rRNA is directly produced from a 5S separate gene

26. Post-transcriptional RNA Processing
B. mRNA – Needs extensive processing
Cap formation (7-methyl-guanylate is added to 5-end)
Poly-adenylation at 3-end (Poly A polymerase)
Splicing
hn RNA
m RNA
(introns + exons)
(exons only)
introns

29. Post-transcriptional RNA Processing
C. tRNA
a. i. One precursor may contain one or more tRNAs
ii. Precursor molecule
Sequences removed
t RNA(s)
b. CCA sequence is added to 3-end
c. Some bases may be modified (i.e. some of A, U, G & C
bases are methylated, reduced & deaminated) before tRNA
can adopt final functional structure