This presentation explains DNA transcription and RNA Processing. It gives details about prokaryotic DNA transcription and eukaryotic DNA transcription. it also explains post-transcriptional modification both in prokaryotes and eukaryotes.

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Asst. Professor
Dept. Of Biochemistry,
SMBT IMS & RC, Nashik
Ashok Katta

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Ashok Katta
Transcription
Learning Objectives
Describe processes of transcription in prokaryotes.
Describe inhibitors of prokaryotic transcription and
their clinical significance
Describe processes involved in transcription in
eukaryotes.
Describe post-transcriptional modifications in
prokaryotes and eukaryotes.
Explain what is reverse transcription & its significance

3. TRANSCRIPTION
It is the process of synthesis of
RNA from DNA as a template.
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Results in the transfer of the information stored
in double stranded DNA into a single stranded
RNA.
Which is used to direct the synthesis of its
proteins.

4. Ribonucleic acid (RNA)
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• Distribution of RNA-
ribosomes & ER
50%
cytoplasm
25%
mitochondria
15%
Nucleus
10%

5. Types of rna
 All are transcribed from DNA.
 The first three RNAs are involved in protein synthesis.
 snRNA is involved in mRNA splicing.

6. Replication Vs Transcription
• Steps of initiation, elongation, &
termination
• Synthesis occurs in the 5’→3’ direction
• Large, multicomponent initiation
complexes
• Follows Watson-Crick base pairing
Similarities
• Ribonucleotides are used rather than
deoxyribonucleotides.
• Uracil replaces Thymine
• Primer is not required
• Only a very small portion of genome is
transcribed
• No proofreading
Differences
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7. • The whole genome of DNA needs to be
replicated, but only small portion of
genome is transcribed in response to the
development requirement, physiological
need and environmental changes.
• DNA regions that can be transcribed into
RNA are called structural genes.
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Transcription Ashok Katta
Transcription in
Prokaryotes

9. Basic requirement for Transcription
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10. Template
A single-strand of DNA acts as a template
The DNA strand that is transcribed to RNA
molecule is referred to as template strand.
The other DNA strand is referred to as the
coding strand of the gene.
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11. Template strand
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DNA
RNA

12. Template strand
• The strand that is transcribed or copied
into an RNA molecule is referred to as
the template strand of the DNA.
• It is also called as…
o Noncoding strand
o Antisense strand
o Anticoding strand
o Transcribed strand
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13. coding strand
• The other DNA strand, the non-template
strand, is frequently referred to as the
coding strand of that gene.
• Because the DNA strand whose base
sequence is identical to the base
sequence of the RNA transcript produced
• It is this strand which contains codons,
while the non-coding strand contains
anticodons.
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14. 14

15. Template strand
• Read out in the 3' to 5' direction.
• The sequence in the RNA is
complementary to the sequence in
template strand of the DNA.
• In the coding strand (complementary
strand) the sequence is same as that of
the sequence of nucleotides in the
primary transcript.

16. Substrates
The four ribonucleoside triphosphate.

17. RNA polymerase

18. ω
σ
Structure of
RNA polymerase (RNAP)
Prokaryotic RNA polymerase
β β’
αα

19.  RNA polymerases lack proofreading
(3′ to 5′ exonuclease activity) as that of
DNA polymerases.
 RNA polymerase requires Mg2+ as well as
Zn2+ for its activity.
RNA polymerase (RNAP)
Rifampicin, a therapeutic drug for
tuberculosis treatment, can bind specifically
to the β subunit of RNA-pol, and inhibit the
RNA synthesis.

20. Transcription unit
• Region of DNA that includes the structural
genes & the signals for initiation,
elongation & termination of transcription.
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21. Recognition of Origins
• Each transcriptable region is called operon.
• One operon includes several structural
genes and upstream regulatory regions.
• The promoter is the DNA sequence that
RNA-pol can bind.
• It is the key point for the transcription
control.
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22. Stages of Transcription

23. Initiation

24. Initiation
Initiation of transcription involves binding of
RNA polymerase (core enzyme + σ factor) to
DNA template at promoter site.
The sigma factor facilitates the RNA
polymerase to recognize and bind to promoter
sequences.
Promoters are characteristic sequences of
DNA which are different in prokaryotes &
eukaryotes

25. Prokaryotic genes have two promoter
sequences
1.Pribnow box (–10 region) has the nucleotide
sequence TATAAT, found 10 base pairs away
(upstream) from the start point.
2. –35 region, has nucleotide sequence
TTGACA, found 35 base pairs away from
(upstream) the start point.

26. Prokaryotic promoter sites for
transcription
3’
5’Structural gene

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28. 3’
5’
σ
Recognition of promoter by sigma factor

29. promoter sequences
 It directs RNA polymerase to initiate
transcription at a particular point known as
initiation site.
 The binding of the RNA polymerase to the DNA
template results in unwinding of DNA double
helix.

30. Termination region
DNA template
RNA polymerase
3’
5’
5’
3’ 5’ σ
Binding of core enzyme starts synthesis of RNA

31. Termination region
DNA templateRNA polymerase
3’
5’
5’
3’
σ
5’
Dissociation of σ-factor

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33. The first nucleotide (NT) on RNA transcript is
always purine triphosphate. (GTP is more
often than ATP.)
GTP (pppGpN-OH) remains on the RNA
transcript until the RNA synthesis is
completed.
The three molecules form a transcription
initiation complex.
RNA-pol (α2ββ’ω) - DNA - pppGpN- OH 3’
No primer is needed for RNA synthesis.

34. RNA polymerase then begins to synthesize a
transcript of DNA sequence and short piece
of RNA is made.
By the time 10 NTs have been added, the
σ-factor dissociates.
The core enzyme moves along the DNA
template to enter the elongation phase.

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Elongation

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Termination regionPromoter region
DNA template
3’
5’
5’
3’
Elongation
Termination regionPromoter region
DNA template
3’
5’
5’
3’
5’
5’

37. Elongation
 Sigma is released and the RNAP move along
the template stand and continues the
elongation of the transcript.
 The process of elongation of the RNA chain
continues until a termination signal is reached

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Termination

39. Termination
In prokaryotes, termination of transcription
occurs by one of the two mechanisms:
1. Rho-dependent
2. Rho-independent.

40. Newly synthesized RNA
Termination region
Promoter region
3’
5’
5’
3’
ρ
3’5’ ρ
Termination of transcription by Rho factor
Rho
factor

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Termination of transcription by Rho factor

42. Rho-independent termination

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• The termination signal is a stretch of 30-40 NTs
on the RNA transcript, consisting of many GC
followed by a series of U.
• Due to this sequence, there is formation of stem-
loop structures on nascent RNA transcript.
• The stem-loop structure alters the conformation
of RNA-pol, leading to the pause of the RNA-pol
moving.
• Finally this causes terminate the transcription.

45. Post-transcriptional Processing
 The RNAs formed during transcription are
called primary transcript.
 The primary transcript normally undergo
further enzymatic alteration, called post-
transcriptional processing.
 Post-transcriptional processing is required to
convert the primary RNAs into functional or
active forms.

46. Processing may involve either:
Post-transcriptional Processing

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Transcription Ashok Katta
Transcription in Eukaryotes

49. Synthesis of rRNARNA
Polymerase I
Synthesis of m-RNA and
snRNA
RNA
Polymerase II
Synthesis of tRNARNA
Polymerase III
Synthesis of all three types
of RNA in mitochondria
Mitochondrial
RNA polymerase 52
Eukaryotic RNA polymerase

50. RNA polymerase of eukaryotes
Products
Sensitivity
to
Amanitin
RNA-pol
I
5.8S,
18S, 28S
rRNA
No
RNA-pol
II
hnRNA,
snRNA
High
RNA-pol
III
tRNA,
5S rRNA
Moderate
Amanitin is a specific inhibitor of RNA-pol. From mushroom.
Inhibits Pol II and Pol III at higher concentrations.

51. eukaryotic promoters
• Eukaryotic promoters are complex, and
composed of several different elements.
• For different RNA polymerase different
promoter complexes are required.

52. RNA polymerase I promoter
rInr - Ribosomal initiator element
UPE - an upstream promoter element

53. RNA polymerase II promoter
Inr - Initiator element
UPE - an upstream promoter element
DPE - downstream promoter element

54. RNA polymerase III promoter

55. RNAP II requires many other proteins called
transcription factors, (TFII) to form the active
transcription complex.

56. stages
The process of transcription by RNAP II can be
described in terms of following stages:
1. Assembly
2. Initiation
3. Elongation
4. Termination

57. TFIIB, TFIIE, TFIIH
TBP TFIIA
TFIIF – RNAP-II
Initiation complex (Open)
Preinitiation complex (closed)
Assembly
1
• Inr: initiator sequences;
• RNAP II: RNA polymerase II;
• TBP: TATA-binding protein;
• TF: transcription factor

58. Initiation complex (Open)
Elongation complex
TFIIE, TFIIH
Initiation
2
Termination complex
Elongation factors
Elongation
3

59. Termination complex
Transcript
Termination factors
Termination
4
Elongation factors
RNAP II
Termination factors

60. Eukaryotic
Post-transcriptional
Processing

61. m-RNA processing
 In eukaryotes the primary transcript of
mRNA is hnRNA.
 After transcription hnRNA is extensively
modified to form functional mRNA.
 Example of these are…
 a cap at near 5’ ends &
 a poly (A) tail at their 3’ ends.
 splicing to remove introns.

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63. 5'-capping
The 5' end of mRNA is capped with 7-
methylguanylate.
The 5′ cap protects the nascent mRNA
from degradation and assists in
ribosome binding during translation.

64. Addition of poly A tail
The 3' end of most eukaryotic mRNAs posses a
chain of 200–300 adenine nucleotides and
called Poly A tail.
Functions:
– Protects the mRNA from degradation,
– Aids in the export of the mature mRNA to the cytoplasm.
– Is involved in binding proteins involved in initiating
translation.

65. Removal of introns
 The process by which introns are excised
and exons are linked to form functional
mRNA is called splicing.

66. Alternative splicing
hnRNA

67. Example of Alternative splicing

69. t RNA Processing
Transfer RNA precursors are converted into mature
tRNAs by following alterations:
1. Cleavage of a 5′ leader sequence
2. Replacement of the 3′ terminal UU by CCA
3. Modification of several bases and sugars
4. Splicing to remove intron

70. t RNA Processing

71. r-RNA processing
• The rRNA precursors called pre-ribosomal
RNAs are cleaved and trimmed to produce
the mature functional r-RNAs.
• Eukaryotic ribosomal RNA processing is
very similar to that of prokaryote.

72. r-RNA processing

73. RNA
Editing
 A change in the base sequence
of RNA after transcription by
process other than RNA
splicing is called RNA editing.
 The process may involve the
insertion, deletion or
substitution of nucleotides in
the RNA molecule.

75. REVERSE TRANSCRIPTION

76. REVERSE TRANSCRIPTION
Certain RNA viruses carry an RNA-
dependent DNA polymerase called
reverse transcriptase.
The RNA viruses that contain reverse
transcriptases are known as
retroviruses
Reverse transcriptase produces
DNA from viral RNA.

77. Extension of the central dogma

78.  Example of retroviruses includes human
immunodeficiency virus (HIV), which causes
acquired immunodeficiency syndrome (AIDS).
REVERSE TRANSCRIPTION

79. Long Answer questions (lAQs)
1. Describe transcription mechanism in eukaryotes under
the following headings:
i. Initiation 3
ii. Elongation 3
iii. Termination 3
iv. Name inhibitors of the process 1
2. Describe post-transcription mechanism in eukaryotes
under the following headings:
i. mRNA processing 6
ii. tRNA processing 2
iii. rRNA processing 2
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80. Brief Answer Questions (BAQs)
1. What is the role of RNA polymerase? Write
component of prokaryotes RNA polymerase.
2. What is transcription? Name the stages of
transcription.
3. Write inhibitors of prokaryotes RNA
synthesis.
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81. short Answer Questions (sAQs)
1. Name the inhibitors of RNA synthesis in prokaryotes
with their clinical significance.
2. Write differences between prokaryotic and eukaryotic
transcription.
3. Write prokaryotic post-transcriptional RNA
processing.
4. What is reverse transcription? How it works?
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82. Reference
• Biochemistry 5th edition by Pankaja Naik
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