The process by which an RNA copy of a gene is made or it’s a DNA dependent RNA synthesis. Transcription resembles replication In its fundamental chemical mechanism Its polarity (direction of synthesis) Its use of a template Transcription differs from replication It does not requires a primer It involves only limited segments of a DNA molecule Within transcribed segments only one DNA strand serves as a template for synthesis of RNA.

1. TRANSCRIPTION
(DNA DEPENDENT RNA SYNTHESIS)
Ms. M. Arthi, M.Sc., M.Phil., NET., SET.,
Assistant Professor
Department of Microbiology

2. CENTRAL DOGMA OF LIFE

3. CENTRAL DOGMA OF LIFE
GENOME: The total DNA (genetic information) present in an organism or a cell.
• Chromosomes in the nucleus
• DNA in mitochondria, and chloroplasts.
Genomics : The study of structure and function of genome is genomics.
 Functional genomics - gene expression and relationship of genes with gene products.
 Structural genomics - structural motifs and complete protein structures.
 Comparative genomics - comparative gene function and phylogeny.
 Metagenomics - genomes of whole communities of microscopic life (microorganisms, viruses).
TRANSCRIPTOME: The RNA copies of the active protein coding genes represent transcriptome
Transcriptomics : The study of transcriptome that involves all the RNA molecules made by a cell,
tissue or an organism is transcriptomics.
PROTEOME: The total proteins and their biological functions in a cell.
Proteomics : The study of the proteome.
Metabolomics : The use of genome sequence analysis for determining the capability of a cell, tissue or
an organism to synthesize small molecules (metabolites) is metabolomics.

4. TRANSCRIPTION
 The process by which an RNA copy of a gene is made or it’s a DNA
dependent RNA synthesis.
 Transcription resembles replication
 In its fundamental chemical mechanism
 Its polarity (direction of synthesis)
 Its use of a template
 Transcription differs from replication
 It does not requires a primer
 It involves only limited segments of a DNA molecule
 Within transcribed segments only one DNA strand serves as a
template for synthesis of RNA.

5.  One strand of DNA - template or non-coding strand or antisense strand -
produces working copies of RNA molecules. The strand that serves as
template for RNA synthesis is called the template strand.
 Other DNA strand – non-template strand or coding strand or sense
strand. The DNA strand complementary to the template, the nontemplate
strand, or coding strand, is identical in base sequence to the RNA
transcribed from the gene, with U in the RNA in place of T in the DNA

6. Transcription is selective
 The entire molecule of DNA is not expressed in transcription.
 RNAs are synthesized only for some selected regions of DNA.
 The product formed in transcription is primary transcript
(Inactive).
 Mature RNA (active) - they undergo certain alterations known as
post-transcriptional modifications (splicing, terminal additions,
base modifications etc.)

7. RNA POLYMERASE
• RNA is synthesized by ‘’RNA polymerase’’, also called as DNA
dependent RNA polymerase.
Discovery of RNA Polymerase
• 1960 – 61 – discovered in animals, plants and bacteria
• 1969 – Richard Burgess and Andrew Travers separated the
polypeptides (subunits) that make up the E. coli RNA polymerase
using SDS-PAGE.

8.  Requirements :
 DNA template
 All 4 ribonucleoside 5’ triphosphates as precursors (ATP, GTP, CTP, UTP)
 Mg 2+
 Zn 2+
 The chemistry and the mechanism is similar to that of DNA polymerase
 It elongates an RNA strand by adding ribonucleotide in 5’----- 3’ direction.
 The product formed in transcription is called primary transcript.
 RNA polymerase lacks proof reading 3’----5’ exonuclease activity hence error
rate is higher when compared to Replication.

9. • Initiation occurs when RNA polymerase binds at specific DNA
sequences called promoters.
• DNA duplex must unwind over a short distance, forming a transcription
“bubble” (17 bp unwound).
• The 5’-triphosphate group of first residue in a nascent (newly
formed) RNA molecule is not cleaved to release PPi, but instead
remains intact throughout the transcription process.
• During the elongation phase - the growing end of the new RNA strand
base-pairs temporarily with the DNA template to form a short hybrid
RNA-DNA double helix (8 bp long) - RNA “peels off” shortly after its
formation from the hybrid duplex and DNA duplex re-forms.

10. • E. coli RNA polymerase – synthesis rate - 50 to 90 nucleotides/s.
• Movement of a transcription bubble requires considerable strand
rotation of the nucleic acid molecules.
• DNA strand rotation is restricted by DNA-binding proteins and other
structural barriers.
• As a result, a moving RNA polymerase generates waves of positive
supercoils ahead of the transcription bubble and negative
supercoils behind
• The topological problems are relieved by the action of
topoisomerases.

12. STRUCTURE OF E. COLI POLYMERASE
• Molecular weight - 390,000 – α2ββ’ω + σ
• It’s a large, complex enzyme with 5 core subunits and sixth sigma subunit.
• These six subunits constitute the RNA polymerase holoenzyme.
• Function – forms an RNA polymer from ribonucleoside 5’ triphosphates
Subunits Molecular mass (kD) Functions
Beta 150 Chain initiation and
elongation
Beta prime 160 DNA binding
Alpha (2) 40 Chain initiation and
interaction with regulatory
proteins
Omega 10
Sigma 70 Promotor recognition

14. • E. coli RNA pol. Exist in several forms, depending on the type of sigma subunit.
• The most common subunit is 70 (Mr 70,000),

15. Cellular RNA polymerases in all living organisms are evolutionary
related
a common structural and functional frame work
of transcription in the three domains of life
LUCA-Last universal common ancestor
Subunits
of
RNAP
Multisubunit RNA polymerases are conserved among all organisms

16. Structure of RNAP in the three domains
Werner and Grohmann (2011),
Nature Rev Micro 9:85-98
Extra RNAP subunits provide interaction sites for transcription
factors, DNA and RNA, and modulate diverse RNAP activities
Universally conserved
Archaeal/eukaryotic
Bacteria Archaea Eukarya
Transcription

18. TRANSCRIPTION MECHANISM
• Transcription is selective – entire DNA molecule is not expressed. RNA is
synthesized only for some selected regions of DNA.
• The product formed in transcription is called Primary transcript.
• The primary RNA transcripts are inactive. Undergo certain alterations like
splicing, terminal addition, base modification etc. commonly called post
transcriptional modifications, to produce active RNA molecules.
• The process involves 3 stages
• Initiation
• Elongation
• Termination

19. A. INITIATION
• RNA polymerase binds to specific sequences in the DNA called promoters.
• RNA polymerases cannot initiate transcription on their own. In bacteria σ70 is required to
initiate transcription at most promoters.
• E. coli RNA polymerase holoenzyme, (core + σ) finds promoter sequences by sliding
along DNA this behavior greatly speeds up the search for specific DNA sequences in the
cell.
• It recognizes two consensus base sequences on the coding DNA strand
• -10 sequences - Pribnow box - (5’)TATAAT(3’) – consist of 6nt, located 10 bases
upstream from the transcription start point
• -35 sequences – (5’)TTGACA(3’) - consists of 6nt, located 35 bases upstream
from the transcription start point.
• A third AT-rich recognition element, called the UP (upstream promoter)
element, occurs between positions -40 and -60 in the promoters of certain highly
expressed genes.

21. ‘holoenzyme’
'

KD ~ 10-9 M
+ 
‘core’
}
Can begin transcription
on promoters and can
elongate
}
Can elongate but cannot
begin transcription at
promoters
Transcription initiation proceeds through a series of structural changes in RNA polymerase, σ70
and DNA.
factor is required for bacterial RNA polymerase to initiate
transcription on promoters
'
Initiation factors

23. THE PATHWAY OF TRANSCRIPTION
 First, the polymerase, directed by its bound factor (sigma factor), binds to the promoter.
Once bound to the promoter site, RNA polymerase is able to unwind the DNA without
the aid of helicases.
 A closed complex (in which the bound DNA is intact) and
 an open complex (in which the bound DNA is intact and partially unwound near
the -10 sequence - rich in adenines and thymines, making it easier to break the
hydrogen bonds).
 Second, transcription is initiated within the complex, leading to a conformational
change that converts the complex to the elongation form, followed by movement of the
transcription complex away from the promoter (promoter clearance).
It consists of two
major parts
Binding
Initiation

24. • A region of unwound DNA equivalent to about two turns of the helix
(about 16–20 bases pairs) becomes the “transcription bubble,”
which moves with the RNA polymerase as it proceeds to transcribe
mRNA from the template DNA strand during elongation.
• Within the transcription bubble, a temporary RNA:DNA hybrid is
formed.
• As the RNA polymerase progresses in the 3′ to 5′ direction
along the DNA template, the sigma factor soon dissociates from
core RNA polymerase.

26. B. ELONGATION
• The σ subunit dissociates as the polymerase enters the elongation phase of transcription.
• The protein NusA (Mr 54,430) binds to the elongating RNA polymerase, competitively with the σ
subunit.
• The mRNA is made in the 5′ to 3′ direction so it is complementary and antiparallel to the
template DNA.
• As elongation of the mRNA continues, single-stranded mRNA is released and the two strands of
DNA behind the transcription bubble resume their double helical structure.
• Once transcription is complete,
• NusA dissociates from the enzyme,
• the RNA polymerase dissociates from the DNA, and
• σ factor (σ 70 or another) can again bind to the enzyme to initiate transcription.

29. TERMINATION
E. coli has at least
two classes of
termination signals
Rho dependent
Rho independent

30. RHO INDEPENDENT TERMINATION
• Most rho-independent terminators have two distinguishing
features.
• The first is a region that produces an RNA transcript with self-
complementary sequences, permitting the formation of a
hairpin structure centered 15 to 20 nucleotides before the
projected end of the RNA strand.
• The second feature is a highly conserved string of three A
residues in the template strand that are transcribed into U
residues near the 3’ end of the hairpin.
• When a polymerase arrives at a termination site with this
structure, it pauses.
• Formation of the hairpin structure in the RNA disrupts several
A=U base pairs in the RNA-DNA hybrid segment and may disrupt
important interactions between RNA and the RNA polymerase,
facilitating dissociation of the transcript.

31. RHO DEPENDENT TERMINATION
• Rho dependent terminators lack the sequence of repeated A
residues in the template strand.
• Synthesized RNA has CA-rich sequence called a rut (rho
utilization) element.
• rho protein associates with the RNA at specific binding sites (rut
site) and migrates in the 5’  3’ direction until it reaches the
transcription complex that is paused at a termination site. It
contributes to the release of RNA transcript.
• rho protein has an ATP-dependent RNA-DNA helicase activity
that promotes translocation of the protein along the RNA, and ATP is
hydrolyzed by rho protein during the termination process.
• The detailed mechanism by which the protein promotes the release
of the RNA transcript is not known.

32. INHIBITORS OF TRANSCRIPTION
The elongation of RNA strands by RNA polymerase in both
bacteria and eukaryotes is inhibited by the antibiotic
• Actinomycin D - inhibits RNA elongation
• Acridine - inhibits RNA synthesis in a similar fashion.
• Rifampicin - inhibits bacterial RNA synthesis by binding
to the β subunit of bacterial RNA polymerases,
preventing the promoter clearance step of transcription.
• α-amanitin (Amanita phalloides) - has a very effective
defense mechanism against predators. It disrupts
mRNA formation in animal cells by blocking Pol I & III.