RNA is synthesized from DNA in a process called transcription. There are three main stages: initiation, elongation, and termination. In initiation, RNA polymerase binds to promoter sequences and unwinds the DNA helix. In elongation, RNA polymerase reads the template strand and adds complementary RNA nucleotides. Termination occurs when termination signals are reached. Prokaryotes and eukaryotes differ in their RNA polymerases and transcription control. Eukaryotic pre-RNA undergoes processing including 5' capping, 3' polyadenylation, splicing, and editing to produce mature mRNA.

1. RNA Synthesis
Surendra Marasini
Lecturer
Department of Biochemistry

2. Overviews
• Introduction
• Transcription in prokaryotes
• Transcription in eukaryotes
• RNA processing (post transcriptional modifications)

3. Central dogma of molecular Biology

4. Introduction
 Synthesis of RNA molecule from DNA – transcription
 Involves a group of DNA dependent RNA polymerase enzymes
and a number of associated proteins
 General steps are initiation, elongation and termination and most
is known about initiation
 Modulation of the transcription
 Error or changes in synthesis, processing, splicing, stability or
function of mRNA transcript are cause of disease

5. Ribosomal RNAs
Transfer RNA
Messenger RNA
Fig: Types of RNA

6. Are the DNA and RNA synthesis similar??
These are similar in following aspects
1. The general steps of initiation, elongation and termination with
5’ to 3’ polarity
2. Large, multicomponent initiation and polymerization
complexes and
3. Adherence to Watson crick base pairing rules

7. What are the differences then???
1. Ribonucleotides are used in RNA synthesis rather than
deoxyribonucleotides
2. U replaces T as a complementary base for A in RNA
3. A primer is not involved in the RNA synthesis
4. A portion of genome is transcribed during RNA synthesis but
entire genome must be copied during replication
5. No highly efficient proofreading function during transcription

8. RNA synthesis comprises three stages
• Like other polymerization reaction : initiation, elongation and
termination
• RNA polymerase perform multiple function in this process
1. Searches promoter sites eg: E.coli DNA has about 2000
promoter sites in its 4.8ҳ 106 bp genome
2. They unwind the short stretch of DNA to produce single
stranded DNA templates from which the sequence of bases
can be easily read out
3. Select correct ribonucleoside triphosphate and catalyze
formation of phosphodiester bond
4. Detect termination signals , where a transcript ends
5. Interact with activator and repressor proteins that modulate
the rate of transcription

9. Prokaryotic and eukaryotic RNA polymerase
RNAP from Thermus aquaticus RNAP from Saccharomyces cerevisiae

10. Prokaryotic RNA polymerase
• Core enzyme – five peptide
subunits
 Enzyme assemby – alpha and
omega
 5’ -3’ polymerase – beta
 This enzyme lacks specificity
• Holoenzyme- sigma subunit
 enables the RNAP to recognize
promoter region

11. RNA polymerase catalyzes transcription
• Fundamental reaction: phosphodiester bond formation
• Thermodynamically favorable

12. RNAP active site
• Includes two metal ions: Mg2+
• One remains tightly bound to
the enzyme and other ion comes
with the nucleoside
triphosphate and leaves with
pyrophosphate
• Three conserved Asp residues
participate in binding these metal
ions
• RNAP are very large complex
enzymes ( eg: RNAP of E.coli –
5 subunits )
Fig: RNAP active site

13. RNAP continue…
Fig: subunits of RNAP of E.coli α2ββ’w

14. RNAP contd…
• A typical eukaryotic RNAP is large and more complex having
12 subunits
• Mol mass : >0.5 milidalton
• Despite the complexity, the structure has been determined by X
ray crystallography in work pioneered by Roger Kornberg and
Seth Darst
• Polymerization reactions catalyzed by RNA polymerase take
place within a complex in DNA termed a transcription bubble

15. Fig: Transcription bubble
• ds DNA locally
unwound in the
region of approx. 17
base pairs

16. Initiation

17. RNA polymerase holoenzyme complex

18. Bacterial promoter sequences

19. Sigma subunits of RNAP recognize promoter
sites
• To initiate transcription, α2ββ’w core of RNA polymerase must
bind the promoter
• Sigma (σ) subunit made this binding possible by enabling RNA
polymerase to recognize promoter sites
• E. coli has several distinct σ factors for recognizing several
types of promoter sequences in the DNA.
• Released when nascent RNA chain reaches 9-10 nucleotides .

20. RNAP must unwind the template double helix
• Transition from closed promoter complex to open promoter
complex
• Requires unwinding of approx. 17 base pairs (1.6 turns)
• This stage is set for the formation of first phosphodiester bond

21. Elongation
Fig: Transcription bubble

22. Continue...
• RNA chains are formed de novo and grow in 5’ to 3’ direction
• The start site of DNA sequence to be transcribed – denoted as
+1, second one +2, nucleotide preceding start site -1
• These designations refer to the coding strand of DNA .
• Newly synthesized RNA chains carry a highly distinctive tag
at 5’end the first base at that end is either pppG or pppA.

23. Fig: Template and coding strands

24. Elongation mechanism
• Ribonucleoside triphosphate binds to the active site of the RNA
polymerase, directly adjacent to the growing RNA chain.
• Incoming ribonucleoside triphophate forms Watson crick base pair
with the template strand
• 3’ OH group at the end of RNA chain attacks newly bound
nucleotide and forms a new phosphodiester bond

25. Elongation continue…
To proceed to the next step…..

26. RNA polymerases backtrack and correct
errors
• RNA – DNA hybrid can also move in the direction opposite to
the elongation
• Backtracking – less favorable energetically because it breaks
bonds between the base pairs
• Very important for proofreading

27. Termination
• It is as precisely controlled as initiation
• In the termination phase of transcription
1. Phosphodiester bonds ceases
2. Melted region of DNA rewinds
3. RNA- DNA hybrid dissociates
4. RNAP releases the DNA
Two mechanisms of termination
1. Rho( ρ) independent
2. Rho (ρ) dependent

28. Rho (ρ) independent

29. Rho (ρ) dependent

30. Transcription in Eukaryotes
Different types of RNAs in eukaryotes

31. Different types of RNAP in eukaryotes

32. Transcription control regions in eukaryotes

34. Commom eukaryotic promoter elements

35. Initiation
• RNAP II – guided by several
Transcription factors
• Binding of TF IID to TATA box
initiates transcription
• Carboxy terminus domain (CTD)
phosphorylation

36. Processing of Eukaryotic pre RNA
RNAP I produces three ribosomal RNAs

37. RNA polymerase III produces transfer RNA
• Most processed of all
• 5’ leader is cleaved
by RNAse P, 3’
trailer is removed and
CCA is added by
CCA adding enzyme
• Undergo
modifications for
function

38. Termination Mechanism in Eukaryotes
Torpedo model
Allosteric model

39. Inhibitors of transcription
In Prokaryotes
Rifampicin
 Semisynthetic derivative of rifamycin
 Derived from strains of Streptomyces spp

40. Continue…
Actinomycin D
 Acts by slightly different mechanism
 Binds tightly and specifically to the double helical DNA and
thereby prevents it from being an effective template for RNA
synthesis
 It does nit bind to ssDNA or RNA, dsRNA or RNA – DNA
hybrids
 Phenoxazine ring of actinomycin slips in between neighboring
base pairs in DNA.

42. RNA polymerase poison in eukaryotes
• α amanitin: cyclic octapeptides that contains several modified
amino acids
• Produces by poisonous mushroom Amanita phalloides
• death cup or destroying angel
• More than 100 deaths per year due to poisonous mushroom
• Binds very tightly (Kd = 10nM) to RNA polymerase II
• Blocks the elongation phase
• Higher concentrations (1 uM) inhibit polymerase III

43. RNA processing
mRNA processing
1. 5’ capping
2. Polyadenylation
3. RNA editing
4. RNA splicing

45. 5’ capping
• Most extensively modified transcription product – product of RNA
polymerase II
• Immediate product of transcription – pre mRNA
• Spliced to remove introns
• Both 5’ and 3’ ends are modified to become mature RNA
• As in prokaryotes, eukaryotic transcription begins with A or G
• However 5’ end of the nascent RNA chain is immediately
modified
• A phosphate is released by hydrolysis then attacks the alpha
phosphorus atom of GTP to form a very unusual 5’-5’triphosphate
linkage

47. Polyadenylation of a primary transcript
• Pre mRNA is also modified at 3’end
• Most eukaryotic mRNAs contain a polyadenylate, poly (A) tail
at the end
• Nucleotide preceding poly A is not last nucleotide to be
transcribed
• Some primary transcripts contain hundreds of nucleotides
beyond the 3’ end of the mature RNA
• How is the 3’ end of pre mRNA given its final form?
 Specific endonucleases – recognizes AAUAAA
 Presence of AAUAAA in the mature RNA indicates that
AAUAAA is only part of the cleavage signal

48. Continue….
• After cleavage by endonuclease a poly A polymerase adds about
250 adenylate residues to the 3’ end of the transcript.
• ATP-donor of this reaction

49. Polyadenylation of primary transcript

50. Functions of 5’cap and 3’ poly A tailing
Functions of 5’capping
 5’cap binds to the cap binding complex, participates in binding
mRNA to ribosome to initiate translation
 Helps to stabilize mRNA
 Prevents the attack from 5’ to 3’ exonuclease ( Ribonuclease)
Functions of poly A tailing
 Stabilize mRNA
 Prevents attack of 3’ to 5’ exonuclease (Ribonuclease)
 Facilitate their exit from nucleus
 Poly A tail & its binding protein (PAB-1) are required for
efficient initiation of protein synthesis

51. RNA Editing
• Changes the proteins encoded by mRNA
• Sequence content is altered after transcription
• Change in the base sequence of RNA after transcription by method
other than splicing
• It is prominent in some systems eg: apolipoprotein B (ApoB)
• ApoB exist in two forms –
• ApoB100
 512kd
 4536 residues
 Synthesized in liver & transport lipids synthesized in cell
• 240kd apoB48
 2152 N terminal residue of ApoB 100
 Synthesis – small intestine, carries – dietary fat in form of
chylomicrons

52. RNA editing continue…
• Truncated molecule – can form the lipoprotein particle –but
cannot bind to the LDL receptor on cell surfaces.
• What is the Biosynthetic relation of these two forms of ApoB?
 One possibility – apo B 48 is produced by proteolytic cleavage
of apoB100
 Another possibility – two forms arise from alternative splicing
(the results of experiments show that neither occurs)
• A totally unexpected and new mechanism for generating diversity:
The changing of a nucleotide sequence of mRNA after its
synthesis
 a specific cytidine residue is deaminated to uridine, whixh changes
the codon at residue 2153 from CAA (Gln) to UAA (stop)

54. Splicing
• Does the particular sequence denotes the splice site?
• Base sequence of thousands of intron –exon junctions within
RNA transcripts are known
• From yeast to mammals, these sequences have a common
structural motifs: the base sequence of an intron begins with GU
and ends with AG
• Consensus sequence at the 5’ splice in vertebrates –
AGGUAAGU
• At 3’end of the intron – consensus sequence is a stretch of 10
pyrimidines (U or C) followed by any base and then by C and
ending with the invariant AG

56. Splicing of nascent mRNA
• Complicated process
• Requires cooperation : small RNAs + proteins = spliceosome
• Two transesterification reaction
• 5’ splice site is attacked by 2’-OH group of the branch site
adenosine residue
• 3’ splice site is attacked by newly formed 3’ OH group
• Introns are released & exons are joined in the form of Lariat.

57. Continue…
Splicing mechanisms used for mRNA precursors

58. Small nuclear RNAs in spliceosomes catalyze
the splicing of mRNA precursors
• Nucleus contains many types of small RNA molecules with
fewer than 300 nucleotides – snRNA (small nuclear RNA)
• Few of them – U1, U2,U4, U5 and U6 -essential for splicing of
mRNA precursors
• Associated with proteins – to form snRNP ( small nuclear
Ribonucleoprotein Particles) “snurps”
• Spliceosomes - composed of other proteins and splicing factors

60. Spliceosome assembly

61. Splicing catalytic center

62. Alternative splicing of some pre mRNA molecules
• Widespread mechanism for generating protein diversity
• RNA products of 30% of human genes are alternatively spliced
• Proteins exhibiting alternative splicing : Actin, alcohol
dehydrogenase, calcitonin
Calcitonin gene related protein

64. Self splicing
• First revealed in 1982 – splicing mechanism of group I rRNA
intron from ciliated protozon Tetrahymena thermophila -
conducted by Thomas Cech & colleagues
• Transcribed Tetrahymena DNA ( including introns) in vitro
using bacterial RNAP
• Resulting RNA spliced itself accurately without any proteins of
Tetrahymena
• Discovery that RNA could have catalytic functions

66. Summary

68. References
1. Biochemistry, Lubert Stryer, 7th Edition
2. Textbook of Biochemistry, Lippincott,
3. Harper’s textbook of Biochemistry, 31st edition