1) RNA is synthesized by RNA polymerase in a process called transcription. In eukaryotes, there are three main types of RNA polymerases that synthesize different RNAs. 2) The basic steps of transcription are initiation, elongation, and termination. In prokaryotes, transcription initiation involves the binding of RNA polymerase and sigma factor to promoter sequences. 3) Eukaryotic transcription is more complex, involving chromatin remodeling and many transcription factors that help recruit RNA polymerase to specific gene promoters. Enhancer sequences can also increase transcription initiation from distant sites on the DNA.

1. RNA Synthesis (Transcription)
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2. Ribonucleic acid (RNA)
• Ribonucleic acid (RNA) is a polymer of purine
and pyrimidine ribonucleotides linked together by
3',5'-phosphodiester bonds analogous to those in
DNA.
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3. Differences between DNA and RNA
1. In RNA, the sugar moiety is ribose rather than the 2'-
deoxyribose of DNA
2. Instead of thymine, RNA contains the ribonucleotide of
uracil.
3. RNA typically exists as a single strand, whereas DNA
exists as a double-stranded helical molecule.
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4. 4. Since the RNA molecule is a single strand, its guanine
content does not necessarily equal its cytosine content, nor
does its adenine content necessarily equal its uracil
content.
5. RNA is easily hydrolyzed by alkali because of presence of
hydroxyl group on 2 carbon atom of ribose.
• Note: RNase are RNA digesting enzymes and DNase are
DNA digesting enzymes.
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5. Types of RNA
• There are mainly three types of RNAs in all prokaryotic
and eukaryotic cells:
Messenger RNA or m-RNA
Transfer RNA or t-RNA
Ribosomal RNA or r-RNA
• They differ from each other by size, function and
stability.
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6. Messenger RNA
• It accounts for 1-5% of cellular RNA.
• Structure: Each mRNA molecule contains a nucleotide
sequence that is converted into the amino acid sequence
of a polypeptide chain.
• mRNAs are the most distinctive class of RNAs when
prokaryotic and eukaryotic cells are compared .
• This is largely because a single prokaryotic mRNA can
encode multiple proteins (it is polycistronic).
• whereas a single eukaryotic mRNA carries the
information to encode only a single protein (it is
monocistronic).
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7.  Other most striking difference between prokaryotic and
eukaryotic mRNAs is that eukaryotic mRNAs are
synthesized as large precursors that have to be
processed before they are functional.
 This processing usually involves the removal of portions
of the transcript, called introns (intervening sequences),
and ligation of the remaining sequences, called exons
(expressed sequences), to one another.
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8. Ribosomal RNA
• Ribosomal RNA or r-RNA accounts for 80% of total
cellular RNA.
• It is present in ribosomes.
• In ribosomes, r-RNA is found in combination with
protein.
• It is known as ribonucleoprotein.
• The length of r-RNA ranges form 100-600 nucleotides.
• Both prokaryotic and eukaryotic ribosomes contain r-
RNA molecules.
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9. Transfer RNA
• t-RNA accounts for 10-15% of total cell RNA.
• During protein synthesis, t-RNA molecules carry amino
acids to ribosomes and ensure that they are incorporated
into the appropriate positions in the growing polypeptide
chain.
• This is done through base-pairing of three bases of the t-
RNA (the anticodon) with the three base codons within
the coding region of the mRNA.
• t-RNA molecules are rather small compared with both
mRNA and the large r-RNA molecules.
• All t-RNA molecules can form a structure resembling a
cloverleaf.
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10. Transcription
• Expression of the information in a gene generally involves
production of an RNA molecule transcribed from a DNA
template.
• RNA is synthesized from a DNA template by the enzyme
RNA polymerase (RNAP).
• Transcription exhibits three phases: initiation, elongation,
and termination.
• Initiation of transcription involves binding of the RNA
polymerase to the promoter region (nucleotide sequence
at the beginning of a length of DNA sequence that is to be
transcribed).
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11. Cont….
• Elongation involves RNA polymerase copying one
strand of the DNA double helix, pairing C's with G's and
A's (on the DNA template) with U's on the RNA
transcript.
• Termination : DNA contains specific sites at which
transcription is terminated.
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12. Transcription cont.…
- The genetic master plan of an organism is contained in DNA.
- RNA “working copy” of DNA (where the plan is expressed).
- Transcription is the copying process, in w/c DNA strand serves
as a template for the synthesis of RNA.
 Transcription produces: mRNAs = translated to AAs,&
- r-RNAs, t-RNAs & other small RNA molecules[aren’t
translated= non coding RNAs (ncRNAs)].
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13. Cont…
- Final product of gene expression is protein or RNA.
- Transcription is highly selective as compared to
replication (all regions of DNA won’t be transcribed).
- This selectivity is due to a guide that instruct RNA
Polymerase where to start, how often to start & where to
stop.
- Regulatory proteins also involve in selection process.
- Transcription is in contrast to the “all or none” of
replication.
- Post transcriptional modification is its typical feature.
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14. Cont…
- inactive 1o transcript functional form by modification.
 RNA Polymerase: catalyze transcription.
a) In bacteria, only 1-type of RNA Pol (multimeric protein) that
synthesize mRNA, t-RNA & r-RNA.
b) In Eukaryotes: Several types:-RNA Pol.I,II & III.
- Coding/sense strand & non-coding/anti-sense strand.
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15. DNA strands used in transcription
– Of the two strands of DNA, one of them is the
template for RNA synthesis of a particular RNA
product.
– RNA polymerase reads it from 3' to 5'.
– Other name is the template strand, because it is the
strand that directs the synthesis of the RNA.
– It is also called the antisense strand, because its code
is the complement of the RNA that is produced.
– It is sometimes called the (-) strand by convention.
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16. – The other strand is called the coding strand because
its sequence of DNA will be the same as the RNA
sequence that is produced (with the exception of U
replacing T).
– Other name is sense strand, because the RNA
sequence is the sequence that we use to determine what
amino acids are produced in the case of mRNA.
– It is also called the (+) strand by convention, or even
the non-template strand.
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17. Transcription of Prokaryotic Genes
A) Properties of prokaryotic RNA polymerase.
 Synthesizes all RNAs except primer (by Primase).
 A multi-subunit enzyme
- recognizes promoter region to bind,
- make RNA complimentary copy of DNA &
- recognize the end of transcription.
 RNA is synthesized in 5’-3’ direction.
 It adds G on C, U specifies A instead of T.
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18. Cont…
- The formed RNA is complimentary to the template (antisense)
strand & identical to the coding (sense)strand with U replacing T.
- In DNA both strands can be a template for transcription but for a
given gene only one of the 2 strands has a promoter region.
- Prokaryotic RNA polymerase is a holoenzyme with.
- Core enzyme:4 subunits (a2BB’) & Sigma factor (σ polypeptide).
- it requires dsDNA & sometimes ssDNA as template and
5’-ribonucleoside triphosphate(UTP,ATP,GTP &CTP).
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19. Cont…
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1) Core enzyme:
- Enzyme assembly(2α)
- β’ for template binding
- β has 5’-3’ RNA polymerase
activity
- Ω its function is unclear
2) Sigma factor (σ-poly peptide)
- enables RNA polymerase to
recognize promoter regions on
DNA.
- Primary transcript which is the
Initial product of transcription.
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20. Steps of transcription
 3 phases of transcription process:
1. Initiation
2. Elongation &
3. Termination.
20
Antiparallel, complimentary base pairs b/n DNA& RNA.
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21. 1.Initiation
- After the σ-factor recognizes the promoter region the holo-
enzyme binds to it trans.complex initiate transcription.
- Prokaryotic promoter region has consensus sequence(20-200
bases).
Chxs of a promoter sequence:
a. –35 sequence: 5'-TTGACA-3',centered 35 bases to the left of the
transcription start site.
- the initial point of contact for the enzyme  closed complex.
- Regulatory sequences: control transcription& found on non
template strand. 21
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22. - -Ve number before a base of a promoter region indicates, it is
found to the left/upstream/(closer to 5’ end)of transcription start
site.
- Therefore, the TTGACA sequence is centered at –35.
- The 1st base at the transcription start site is assigned as +1.
b. Pribnow box:-Named after David Pribnow/TATA box
- The 2nd consensus sequence(5'-TATAAT-3') spanned by the
holoenzyme,centered at –10,w/c is the site of initial DNA melting
(unwinding).
- The T at position 6 is present in any promoter region; so it is
conserved. 22
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23. 23
Local unwinding of DNA by RNA polymerase and formation of an open initiation
complex.
Structure of the prokaryotic promoter region.
Melting of a short stretch (~14 bases) converts the closed complex to an open
one a transcription bubble.
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24. 2. Elongation:
- When the holoenzyme unwinds DNA supercoils(DNA-
topoisomerase overcome this problem).
- RNA polymerase begins to synthesize a transcript of the DNA
sequence.
- The elongation phase begins when the transcript (typically
starting with a purine) exceeds 10 ntds in length.
- σ-is then released, & the core enzyme proceeds processively.
- RNA pol: Substrates (ATP,GTP,CTP,UTP) & releases PPi .
- As replication, transcription is always in the 5'→3' direction.
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25. Cont…
- When about 10 nucleotides have been incorporated, the -subunit
dissociates and is later recycled to bind to another RNA
polymerase core enzyme.
- Unlike DNA pol, RNA pol doesn’t require a primer & have no
proofreading activity.
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3. Termination:
- ssRNA elongates until a termination signal is reached.
- 2 types of terminations
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26. Cont…
1.ρ-independent/intrinsic/spontaneous: seen prokaryotic genes
- The nascent RNA-generates a sequence i.e. self complimentary.
- RNA folds back on itself GC-rich stem & loop “hairpin”.
- beyond the hairpin, the RNA transcript contains a string of ‘U’s at the 3'-
endbonding with DNA’s ‘A’s weak linkage facilitate easy separation
of RNA from its template as the double helix “zips up”behind the RNA pol.
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27. Cont…
2) ρ-dependent:-help of rho(p) protein/requires ATP
- binds a C-rich “rho recognition site” near the 3'-end of the nascent RNA &
chases RNA polymerase along RNA force it to pause.
- The ATP dependent helicase activity of ρ-separates the RNA-DNA hybrid
helix the release of the RNA.
NOTE: some antibiotics work by inhibiting RNA synthesis.
- e.g.,Rifampin(anti-TB):inhibit initiation by binding with β-subunit of prok
RNA poly conformational change.
- but rifampin doesn’t bind to Eukaryotic RNA polymerase. Why?
2. Actinomycine D: used in tumor chemotherapy & works by binding with
DNA template and affect movement of RNA pol.
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28. II. Transcription of Eukaryotic Genes
- More complicated than in prokaryotes.
- Involves separate RNA pols.to synthesize r-RNA, t-RNA& mRNA.
- Also, a large number of proteins called TFs are involved.
- TFs bind on the DNA in promoter region, close to it, or distal.
- TFs are needed for the assembly of a trans.complex at the promoter &
determine w/c genes are to be transcribed.
- Each eukaryotic RNA polymerase has its own promoters & TFs.
- for TFs to recognize & bind to promoter, the chromosome must be
remodeled to increase access.
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29. Cont…
A. Chromatin structure & gene expression
- Euchromatin: relaxed and active form to be transcribed.
- Inactive: in condensed form= Heterochromatin
- The interconversion b/n the 2 forms =Chromatin remodeling.
 Formation of Nucleosome affects transcription.
- Acetylation & deacetylation of Lys residue of histone proteins at
N-terminus mediate remodeling.
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30. Cont…
1. Acetylation of Lys residue by Histone acetyl transferase (HATs)
eliminates the +Ve charge on Lys &↓es interaction of Histone
with –Vely charged DNA increase accessibility.
2. Histone deacetylases (HDACs) remove the acetyl group-favor
strong interaction b/n histone & DNA.
B. RNA polymerases of Eukaryotic Cells: 3 Types.
1.RNA Pol-I: Synthesizes the precursor of 28S,18S& 5.8S r-RNA.
2. RNA Pol-II: Synthesizes precursor of mRNA & certain small
ncRNAs; like, snRNA.
3. RNA Pol-III: synthesizes tRNA,5SrRNA,&some snRNA.
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31. Cont…
a) Promoters & TFs for RNA Pol-II
- ntds are identical with that of Pribnow box but at -25 bases.
- this promoter consensus sequence=TATA or Hogness box.
- CAAT(2nd consensus sequence): at -70 to -80ntds from start site.
- In constitutively expressed genes, no TATA box is present;
instead, a GC-rich region (GC box) may be found.
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32. Cont…
- Consensus sequences are on the same DNA strand to be
transcribed, hence they are called cis-acting elements.
- Such sequences serve as binding sites for TFs, w/c in turn
interact with each other & with RNA pol.II.
- TFs are encoded by d/t genes & synthesized in cytosol, and
act in nucleus; hence, they are called Trans-acting factors.
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33. Cont…
 Function of TFs: Promoter recognition
- Recruitment of RNA polymerase to the promoter& initiate
transcription.
 RNA Polymerase-II doesn’t recognize & bind the promoter by
itself.
a) TF-IID- recognizes & bind to the TATA box.
b) TF-IIF-brings the polymerase to the promoter.
c) TF-IIH: has helicase activity melts the DNA.
- TFs bind outside & inside the promoter sequence to modulate
transcription in response to cell signals like hormones. 33
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34. Cont…
- Some TFs also bind to proteins(“coactivators”),recruiting them to the
transcription complex. Coactivators: like HAT enzymes.
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Eukaryotic gene
promoter consensus
sequences.
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35. b. Role of enhancers
 Enhancers are special Cis-acting DNA sequences that ↑es the
transcription initiation rate by RNA pol-II.
- 1. located upstream/5'-side or downstream/3'-side of start site.
- 2. ~ thousands of base pairs away from the promoter &
- 3. occur on either strand of the DNA.
 Enhancers contain DNA sequences called “response elements”
that bind specific TFs w/c bound to promoter activate RNA-
pol-II.
35
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36. Cont…
 Silencers: act over long distances & reduce gene expression.
 α-Amanitin (toxin from mush rooms)/death cap inhibits RNA-
pol-II tight complex with polymerase  Inhibit mRNA
synthesis.
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Possible locations of enhancers
B. mtRNA polymerase
- Mitochondria contain a single RNA pol.
closely resembles bacterial RNA pol.
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37. 37
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38. V. Posttranscriptional Modification of RNA
- 1o transcript is the initial,linear,RNA copy of a transcription
unit(precursor).
- The precursors of both prokaryotic & eukaryotic t-RNA & r-
RNA are post transcriptionally modified by ribonucleases’
cleavage.
- t-RNAs further modified to give each species its unique
identity.
- In contrast, prokaryotic mRNA is identical to its 1o transcript,
but eukaryotic is extensively modified both co-&post
transcriptionally. 38
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39. A. r-RNA
 Formed from a single precursor pre-r-RNA in both Euk & prokaryotes.
- In prokaryotes:23S,16S & 5S r-RNA.
- In Eukaryotes:28S,18S & 5.8S r-RNA.
- In Eukaryotes 5S is synthesized by RNA Pol III & modified separately.
- Enzymes for modification:ribonucleases (RNases)& exonucleases.
- RNA synthesis & processing occur in the nucleolus, with base and sugar
modifications facilitated by small nucleolar RNAs (snoRNA).
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Posttranscriptional processing of
eukaryotic r-RNA by ribonucleases.
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40. B. t-RNA
 formed from longer precursor in both Euk&prok modified
(post transcriptionally).
- Sequences at both ends of the molecule are removed;
- if intron is present,cleaved by nucleases from anticodon loop.
- a–CCA sequence is added by Nucleotidyltransferase at its 3'-
end,and modification of bases at specific positions to produce
the “Unusual bases” chxs of tRNA.
- Modified bases include D(dihydrouracil),ψ(pseudouracil).
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41. 41
Posttranscriptional modification of tRNA-transcript.
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42. Capping
• The protein-coding genes of eukaryotes are transcribed
by RNA polymerase II to form primary transcripts or
pre-mRNAs that serve as precursors to mRNA.
• These RNA molecules are very large and their
nucleotide sequences are very heterogeneous because
they represent the transcripts of many different genes,
hence the designation heterogeneous nuclear RNA, or
hnRNA.
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43. • Shortly after transcription of hnRNA is initiated, the 5’
end of the growing transcript is capped by addition of a
guanylyl residue.
• This reaction is catalyzed by the nuclear enzyme
guanylyl transferase using GTP as substrate.
• The cap structure is methylated at the 7-position of the
G residue
• This m7 cap, which is added to the growing transcript
before it is 30 nucleotides long, defines the eukaryotic
translational start site and protects the mRNA from
exonuclease degradation.
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44. Polyadenylation
• Most eukaryotic mRNAs have 100 to 200 adenine
residues attached at their 3’ end, the poly(A) tail.
• These A residues are not encoded in the DNA but are
added post transcriptionally by the enzyme poly(A)
polymerase, using ATP as a substrate.
• It is thought that the presence of the tail protects the
mRNA from nucleases and phosphatases, which would
degrade it.
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45. Splicing
• Eukaryotic genes frequently contain intervening
sequences that do not appear in the final base sequence of
the mRNA for that gene product.
• The DNA sequences that are expressed (the ones actually
retained in the final mRNA product) are called exons.
• The intervening sequences, which are not expressed, are
called introns.
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46. • Such genes are often referred to as split genes.
• The formation of mature eukaryotic mRNA involves
removal of introns and linking the exons together in a
process called gene splicing or just splicing
• Splicing occurs when the various snRNPs (small nuclear
ribonucleoprotein) come together with the pre-mRNA to
form a multicomponent complex called the spliceosome.
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47. Alternative Splicing
– In one mode of splicing, every intron is removed and
every exon is incorporated into the mature RNA
without exception.
– This type of splicing, termed constitutive splicing,
results in a single form of mature mRNA from the
primary transcript.
– However, many eukaryotic genes can give rise to
multiple forms of mature RNA transcripts.
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48. 48
Thank You!
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