Transcription is the first step in gene expression. It involves copying a gene's DNA sequence to make an RNA molecule. Transcription is performed by enzymes called RNA polymerases, which link nucleotides to form an RNA strand (using a DNA strand as a template).

1. TRANSCRIPTIONANDPOST
TRANSCRIPTIONALMODIFICATIONS
YASHGUPTA
DEPARTMENTOFBIOTECHNOLOGY ENGINEERING
INSTITUTEOFENGINEERINGANDTECHNOLOGY
BUNDELKHAND UNIVERSITY JHANSI
MOLECULAR BIOLOGY

2. TRANSCRIPTION
• Theprocessof copying information from DNAto RNAiscalled transcription
• Similar to DNA,RNAalsocontains4 different nucleotide baseswhichare linked
by phosphodiester bond
• ButRNAhaveribose sugar backbone inplace of deoxyribose backbone inDNA
and containUracil instead of Thyminealong with Adenine, Guanineand Cytosine
• DNAdependent RNApolymerase isa leading enzymethat mediates this process
• Thisprocessisinitiated with unwinding of double stranded DNA
• One DNA strand behave as template (non-coding or antisense strand) for RNA
synthesisonwhich complementary RNAstrandforms
• Theother DNAstrand called coding or sensestrand hasthesamesequenceasthat of the
formed RNAtranscript but inplace of Uracil inRNAthisstrand containsThymine
Figure1: Componentsof nucleotide (Source:
https://courses.lumenlearning.com/wm-
biology1/chapter/reading-structure-of-
nucleic-acids/)

3. Figure2: Machinery of transcriptiondepicting essentialcomponents(Source:http://rsg-
germany.iscbsc.org/january2019/)

4. DIFFERENTTYPESOFRNASSYNTHESIZEDBYCELL
RNA TYPE FUNCTIONS
mRNA messengerRNA,proteincoding
rRNA ribosomal RNA,form basicribosomal structureand
contribute in translation
tRNA transfer RNA,work asadaptor intranslation
between aminoacid and mRNA
snRNA smallnuclear RNA,function invarious nuclear
processeslike splicing of pre- mRNA
snoRNA smallnucleolar RNAs,contribute in chemical
modification and processing of rRNAs
Several noncoding RNA functionsin variety of cellular processes, including
chromosomalend or telomere synthesis,
inactivation of Xchromosome,and protein transport
into ER

5. PROKARYOTICRNAPOLYMERASE
• RNApolymerase enzyme isa multi subunit complexresponsible for phosphodiester
bond catalysis between two subsequent nucleotides
• It has6 subunitsα2, β, β’, ω,andσ
• It scrollonDNAstep by step and unwindsthedouble helix
• RNAtranscript extendsindirection from 5’ to 3’
• No primer isneeded for to start transcription
• It makesaround 1 error inevery 104 nucleotide transcribed on RNA
• It canproof read, if a wrong baseisadded, theenzymecanback up and active site
excisethenucleotides where incorrect base isadded and synthesize again
Figure3: Exact
polymerase in
conformation of RNA
transcription (Source:
Molecular Biologyof theCell. 4th edition.
Alberts B,JohnsonA,LewisJ,et al.
NewYork:Garland Science; 2002.)
Subunit Mol. Wt. Function
α 40,000 Provideproper structure
β 150,000 Helpsin polymerization
β’ 160,000 AidsRNAPolymerase binding to template
ω 11,000 Provideproper structure
σ 70,000 Promotersequencerecognition and helpsinRNAPolymerase binding to DNAinitiationsite

6. TRANSCRIPTIONINPROKARYOTES
• Transcription initiation occurs when σ70 factor (subunit of polymerase) in combined state with RNApolymerase
encountersthepromoter sequenceinDNAwhile scanning it
• Promotersequenceof DNAcontains2 hexamericsequencesat -35 (TTGACA)and -10 (TATAATGPribnow box)
positions
• It binds to thispromoter or initiation sequencetightly and opensa smallregion of DNAhelix at -10 position (by a
reversible changeinstructure)
• Now onestrand of DNAact astemplate, by complementary base pairing polymerase joinstwo ribonucleotides at first
to initiate RNAchain
• Addition of ribonucleotides continuesthereafter (Untill 10 nucleotides called abortive initiation)
• σfactor dissociates after thechainbecomesaround 10 nucleotide long
• RNApolymerase continueto moveforward without σfactor by undergoing somestructural changes
• Elongation of RNAchainoccursat a speed of 50 ribonucleotides per sec
• When thepolymerase encounterstermination sequenceit dissociates and releasesnewRNAand template DNA
• After release polymerase again associate with σfactor and proceed to searchanother promoter

7. Figure4: Process of transcription in bacteria (Source:
Molecular Biology of the Cell. 4th edition. Alberts B, Johnson
A,LewisJ,et al. NewYork:Garland Science;2002.)
• Somespecial feature of RNApolymerase
• Asthe σfactor placespolymerase oncorrect position at promoter and an
unwoundDNAtemplate hasbeen movedto the active site, the two movable
jaws of polymerase clamponto DNA
• After transcription of ten nucleotide and dissociation of σ factor, a back
flap of polymerase closes and form exit tunnel (RNAleaves through this
tunnel)
• A rudder like polymerase part continuously interferes and keeps the DNA
RNAapart
• Inbacteria thetermination sequenceconsistsof a A-Tnucleotide pairs
string which comesbefore a DNAsequencehaving 2 fold symmetry
• Thissequencewhentranscribed formsa hairpin loop via WatsonCrick
pairing of bases
• This hair pin also help to open RNApolymerase flap and promotes RNA
release from exit tunnel
• Simultaneously,thehybrid DNARNAU-Abasepair dissociates and
result inpolymerase dissociation from DNAand itsrelease by forcing
jaw opening
• When polymerase movesleft to right thebottom strand act astemplate
but whenit movesright to left upper strand moveastemplate

8. TERMINATION
• Termination of transcription occur via
two modes either rho dependent or
rho independent
• Rhoindependent termination occur via
formation of hair pin loop
• Rho dependent uses rho factor for
dissociation of hybrid (DNA-RNA)
along with hair pin loop
Figure5: Rhoindependentand rhodependent termination of transcription(Source: Medicinal
Biochemistry.2nd edition BayneeesJ& DominiczakM.Saunders:Elsevier; 2015.)

9. REGULATIONOFTRANSCRIPTIONIN PROKARYOTES
Figure6:Tryptophan operonshowingregulation of genesin thepresence and
absenceof tryptophan (Source:Pearsoneducation Inc. 2014)
Figure7: Lactoseoperonshowingregulation of genesin thepresenceandabsence
of lactose(Source:Genetics2nd Edition W.H.Freemanand Company2005)

10. TRANSCRIPTIONINEUKARYOTES
• Eukaryotes contain3 RNApolymerasesRNApolymerase I,IIand III
• RNApol I transcribes28S,18S and 5.8S rRNA
• RNApol IItranscribes protein coding mRNAs,snoRNAand few snRNA
• RNApol IIItranscribestRNAs,5SrRNAs,snRNAsand smallRNAgene
• Various transcription factors (TF) are needed by pol II to initiate transcription in
eukaryotes
• Someconsensussequencesare present in DNAwhich initiate transcription
• Promoter in eukaryotes contain TATA box located 25 nucleotide upstream to
initiation site
• TATAbox isrecognized by TFIIDwhich bindsvia TATAbinding protein (TBP)and then
enable TFIIBbinding at this site
• Thenother TFand polymerases assembleonpromoter sequenceof DNAasshown in
figure
• TFIIH employs ATP to open double helix at start site, it also phosphorylate
polymerase IIat Cterminal tail to release it from TFssothat it maystart elongation
Figure4: Transcription initiation by RNA pol II (Source:
Molecular Biology of the Cell. 4th edition. Alberts B,
Johnson A, Lewis J, et al. New York: Garland Science;
2002.)

11. • Some consensus sequences which lie in close vicinity
of RNA polymerase II during transcription are BRE,
TATA,INRand DPE
• As the DNA of eukaryotes is
nucleosomes that are in turn
packed
arranged
into
into
chromatin structures, so to open the DNA some
additional proteinsare needed
• Transcriptional activators bind to specific DNA
Figure5: Consensus sequences of DNA needed for transcription initiation (Source:
Molecular Biology of the Cell. 4th edition. Alberts B,JohnsonA, Lewis J,et al. New
York:Garland Science; 2002.
sequences and attract RNApol IIat start site of
transcription
• Thiseliminates thedifficulty faced by TFand RNA
pol IIto bind DNAwhichispacked inchromatin
• Mediators allows proper communication of
activators withTFand pol II
• Further chromatin remodeling complexesare needed
along with histone acetylase to enhance accessibility
to DNAto initiate transcription
Figure6: Overall assemblyof transcription initiation complex (Source: Molecular
Biology of theCell. 4th edition.Alberts B,JohnsonA,LewisJ,et al. NewYork:
Garland Science; 2002.)

12. • Through out elongation RNA pol II
remain bound to TFIIF
• In elongation, elongation factors
increases activity of polymerase, repress
any pausing and coordinate transcription
complex with post transcriptional
modification proteins
• Elongation factors which mediate these
processes are ELL (eleven nineteen lysin
rich leukemia), p-TEFb, SII (TFIIS), elongin
(SIII)
• After completion of transcript of RNA,
transcription is terminated, polymerase II
is dephosphorylated and whole
assembly dissociates
Figure7: Proteinsrequired for transcriptionin eukaryotes(LehningerPrinciples of Biochemistry.7th
edition. David L.Nelson,MichaelM.CoxEngland:W. H.Freemanand Company; 2017

13. REGULATIONOFEUKARYOTICTRANSCRIPTION
• Additional promoter sequences like CCAAT
box and GC box upstream to TATA box
regulate theinitiation of thymidine kinasegene
transcription
• SV40 promoter sequence have 1 TATA box, 6
GC box and 1 enhancer sequence (72 bp
repeats) for early and efficient transcription
thusregulate transcription
• Specific proteins act as enhancer after binding
specific sequences upstream to initiation site of
transcription
Transcription factors Consensusbinding site
Specificity protein1 (Sp1) GGGCGG
Enhancerbinding protein(EBP) CCAAT
Activator protein1 (AP1) TGACTCA
Octamer binding proteins (OCT-1
and OCT-2)
ATGCAAAT
E-boxbinding proteins (E-12, 47, 2-
2)
CANNTGa
Figure8: Specific promoter or enhancer sequences which regulate
transcription (Source: The Cell: A Molecular Approach. 2nd
edition. Cooper GM. Sunderland(MA): SinauerAssociates; 2000.)
Source:TheCell:AMolecularApproach. 2nd edition. Cooper GM. Sunderland(MA):
SinauerAssociates;2000.

14. POSTTRANSCRIPTIONALMODIFICATIONS
• Thenewly formed transcript containsomenoncodingregions called intronswhichneedsto
be excisedand the coding region called exonsare joined by the processcalled splicing
• Amodification at 5’ end isaddition of 5’ cap and at 3’ end isaddition of poly Atail
(containingaround 80-250 A residues)
• Thesemodifications protect mRNAfrom attack of nucleasesat the end
• 5’ cap play role in initiation of translation after binding with cap bindingproteins,this
complex binds to 40S subunitof ribosome

15. 5’ CAPPINGOFTRANSCRIPT
• After synthesis of 25 nucleotides the nascent
RNA5’ end ismodified by capping
• Thisreaction ismodified by 3 enzymes
• PhosphataseremovesPform 5’end
• Guanyl transferase adds GMP(5’ - 5’ linkage)
• Methyl transferase add CH3group to guanosine
• All three bind to RNApol phosphorylated tail
Figure9: 5’ capping of mRNA(Source: Molecular Biology of the Cell. 4th
edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science;
2002.)

16. SPLICING
• Splicing removes introns from mRNAtranscript and joins
exon to form mature mRNAto be usedfor translation
• Splicing canbe performed with or without formation
of spliceosomes(snRNPsand other proteins)
• In splicing without spliceosomes, one splicing removes
one intron by two subsequent phosphoryl transfer
reactions (transesterifications)
• Thesereactions join 2 exonsand removesoneintronas
• In spliceosome mediated splicingATPisutilized for
step wise assemblage and arrangement of
spliceosomes Figure10: Splicing of intron by lariat formation (Source: Molecular
Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New
York:Garland Science;2002.)

17. Figure11:Splicingof intron by lariat formation mediated by snRNPsand otherproteins forming a spliceosome (Source:
Molecular Biologyof theCell. 4th edition.Alberts B,JohnsonA,LewisJ,et al. NewYork:Garland Science; 2002.)

18. 3’ POLYADENYLATION OF
TRANSCRIPT
• Consensus sequences of nucleotides direct
cleavage and thenpoly Aaddition on3’ end
• Such sequences are present in genome and are
identified after being transcribed in transcript of
RNAby specific proteins
Figure12: Consensus sequences that mediated polyadenylation
(Source: Molecular Biology of the Cell. 4th edition. Alberts B,
JohnsonA, LewisJ,et al. NewYork:Garland Science;2002.)
Figure13: Mechanismof polyadenylation (Source:Molecular Biology of theCell. 4th
edition.Alberts B,JohnsonA,LewisJ,et al. NewYork:Garland Science;2002.)

19. INHIBITORSOFTRANSCRIPTION
• Both prokaryotic or eukaryotic RNApolymerases suffers inhibition by actinomycin
D which intercalates with double stranded DNA and prevents movement of RNA
polymerase on DNA
• Acridine worksin similar mannerand inhibits RNAsynthesis
• Rifamycin binds to β subunitof RNA polymerase of bacteria and inhibit the
promoter clearance transcription step
• α-amanitin blocks animal RNA polymerase II and at high concentration RNA
polymerase III

20. REFERENCES
• Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York:
Garland Science; 2002. From DNA to RNA. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK26887/
• Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer
Associates; 2000. Regulation of Transcription in Eukaryotes. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK9904/
• NelsonDL,CoxMM. LehningerPrinciplesof Biochemistry. 7th edition. England:W. H.
Freemanand Company;2017.