TRANSCRIPTION

• By
• S. Sivaranjani Arunnehru
• Assistant Professor
• Bon secours college for women
• Thanjavur
INTRODUCTION
• Presence of gene in the genome is not sufficient to express as proteins.
• Genes proteins Gene Expression
It is divided into 2 phases
 Transcription or RNA synthesis
 Translation or protein synthesis

Transcription:
 It is the first step in gene expression and is predominant in level of gene regulation
It is another type of Nucleic acid produces another type of it in the process of RNA synthesis
from DNA molecule with the help of complex protein

Concept:
mRNA synthesis Carry information form gene located in nucleus to ribosome to
cytoplasm (Crick, 1958).
BASIC MECHANISM OF TRANSCRIPTION:

• INITIATION:
• RNA polymerase enzyme recognize promotor region lie just “upstream” of gene and bind to it.
• Initial structure formed between RNA polymerase and DNA –closed promoter complex. Enzyme covers or
protect about 6 bp of double helix starts from just upstream of 35 box and down stream of 10 box.
• 10 box the DNA helix unwinds due to breakage of bp [melting]. Melting is an essential prerequisite for
transcription : bases of template strand must be exposed in order to discrete transcription.
• Synthesis of open promoter complex is forced I ST 2 ribonucleotide are base paired to template
polynucleotide at position +1 and +2 and I st phosphodiester bond of the RNA molecule is synthesized by
RNA polymerase.
• Transcription follows same base pairing rules as DNA replication.
• T, G, C, U and A in DNA pair.
• Bp pattern ensures that the RNA transcription is faithful copy of gene.
• End of initiation signified by promoter.
• Clearance, where RNA polymerase moves away from the promoter site without dissociating, freeing the
promoter from further initiation events.
• Promoter clearance occur only if the open promoter complex is stable and follows a number of abortive
initiation where small transcription are generated.
• Initiation –rate limiting steps in transcription and high level of gene regulation in both prokaryote and
eukaryote.
 ELONGATION:

• RNA polymerase moves along the template synthesize nascent RNA in 5-3 direction. The DNA is
unwound ahead of elongation complex and rewound behind dynamic , melting structure represent
the site of transcription – trans bubble.
• Transcription bubble – between 12 to 17 bp in length and is constant through out transcription.
• RNA polymerase – unwindase and windase activity as no other enzymes such as helicase or
topoisomerase taking part in transcription.
• Polymerization- 3’ oh group pf one ribonucleotide reacts with 5’ po4 of 2 nd ribonucleotide to form a
phosphodiester bond- loss of pi for each bond formed.
• The chemical reaction modulated by the presence of DNA template directs the order in individual
ribonucleotide are polymerized into RNA.
• As polymerase move along , it must insert the proper nucleotide into growing chains at each site.the
enzyme is capable of selecting the complementary ribonucleotide triphosphate for incorporation as a
result of ability of nucleotide to form the proper stereo chemical fit with nucleotide in DNA strand being
transcribed .
• Bacterial RNA polymerase is capable of incorporate 50 nucleotide/second into growing RNA.
• New RNA transcribed from 5’ to 3’ direction .
• New ribonucleotide being added to free 3’ end of existing polymer.
• The length varies from gene to gene or it may be short as 20 nucleotide or longer than 600 nucleotides .
TERMINATION:

• It is not a random process, but must occur only at a suitable position shortly after the end of all gene.
• Polymerase moves down to the DNA until RNA polymerase reached a stop signal or terminal sequence.
• Termination occur with the help of rho protein is known as rho depended termination and which occur
without the help of rho protein is called rho- in depended termination.
• Both rho depended and rho in depended termination involve the formation of stem loop.
• RNA polymerase pauses at the stem loop structure, rho catches up to the polymerase and unwind the
DNA RNA hybrid . rho can do it has DNA RNA helicase property. Yet , exact segment of events at the
terminator is not fully known presently.
TRANSCRIPTION IN EUKARYOTES:

• It is a complex process- one group of RNA polymerase and associated proteins are involved in this
process.
• Different RNA polymerase exist for different types of gene.
• Process of eukaryotic transcription is less understood because certain RNAs and mRNAs have different
lifespans in cell complicate the situation and mammalian cell different from prokaryotes.
RNA POLYMERASE:
• There are 3 different types of RNA polymerase:
• RNA polymerase I: nucleolus – transcription of genes for rRNA.
• RNA polymerase II: synthesis of heterogenous nuclear RNA or mRNA.
• RNA polymerase III: transcription of small nuclear RNA and t RNA.
• Size of RNA polymerase- Molecular weight 500,000 to 600,000.
• All eukaryotic RNA polymerase have similar structure and to extent have same AA segment but respond to one
peptide toxin [α amanitin] in different ways.
• Α amanitin blocks the translocation of RNA polymerase during transcription .
• RNA polymerase I insensitive to α amanitin but RNA polymerase II is sensitive even to lower concentration,
where as RNA polymerase III is sensitive at high concentration of α- amanitin.
• Eukaryotic polymerase II consists of 14 subunits .
• Carbonyl terminal repeats domain [CTD] -26 repeated units in yeast and 52 units in mammalian cells .
• Promoters:
• Region in which RNA polymerase binds and begin transcription.
• In eukaryotic , promoters do not start –frequently events to occur.
• 3 different RNA polymerase in eukaryotic nuclei.
• More than 3 different promoters recognized by different polymerase.
• RNA polymerase does not recognize eukaryotic promoters by itself. For the recognition RNA polymerase
require help or relies on proteins and transcription factors.
• Promoters of RNA polymerase II: polymerase II responsible for transcribe the vast majority of gene coding
for proteins.
• Confer fidelity and frequency of initiation and rigid require for both position and concentration . single base
changes have dramatic effect on functions.
• RNA polymerase III: Promoters that governs 5S transcription of 5s rRNA and tRNA genes transcription
different from bacterial polymerase. III promoters located within the genes they enter.
• Promoters of +50 to +83 bp from start point of transcription it is a internal control region.5s RNA gene
promoter split into A and C box with short segment in middle- intermediate element.
• tRNA , ICR split into A and C box without any intermediate element sequence between 2 boxes is of least
important transcribe gene encoding other small RNAs contain promoters and within gene but at 5 end of
gene starting. One small RNA TSL RNA contain 1 TATA box.
• RNA polymerase I: base sequence not match with TATA or CAT box. It recognize promoters present on
rRNA gene.
• Promoters of this gene present at the 5’ end of gene.
• Ist promoters core element between -45 and +20.
• This promoter segment is conserved like TATA box and 65 bp in length.
• Other found between -156 to -107 before transcription site – upstream control element [UCE] 49 bp in
length.
TRANSCRIPTION FACTOR:

• Protein assist RNA polymerase in recognize promoters.

• Two groups:
• Activate specific genes: gene specific transcription factors or gene activation.
• Activate any gene: general transcription [GT] factor
• GT bind DNA region within promoters by themselves and deliver RNA polymerase to promoter site.
• GT occur at low level or basal level but gene specific transcriptase can act both +ve[boost the
transcription rate] or – ve [inhibit transcription rate].
• RNA polymerase I transcription factor:
• Pre initiation complex forms at rRNA promoters involves binding if polymerase I in addition to 2
transcription factors called SL-4 and upstream binding factor [UBF] to the promoter. UBF binds to
upstream control element of rRNA promoters and causes polymerase I to bind near the
transcription start site.
• UBF binds SLI join complex strength polymerase I binding promoter- formation of pre initiation complex.
• SLI – complex protein with a peptide- TBP – one of protein in TATA binding protein family.
• Similarly to TFIID and 3 peptides bind to TBP 3 peptide – TAF3 and similarly to TAF5 in TFIID.
• RNA polymerase III: transcribe different type of genes , transcription complex are also different transcription complex
are same but complex formed are different.
• 3 factors involved in 5s rRNA transcription:
• TFIIA,B AND C
• TFIIB – true transcription factor others are assembly factors. It helps RNA polymerase III binds just down stream in a
position to start transcribe the gene.
• TFIIIB and C bind to internal promoter followed by TFIIIB.
• TFIIIB position RNA polymerase III at transcription start site.
• tRNA gene transcription 2 factors- TFIIIB and C –binds internal promoter. TFIIIC – protein protein interaction.
• TFIIIB contains TBP like S1 and same role- SL1.
• TFIIA and C – Same role as UBF.
• RNA polymerase trans factors: it is a complex interact with many factors before they initiate the
transcription.
• 3 classes of transcription factors- regulation of transcription by RNA polymerase II.
• Ist class –basal factors . Ex: TBP, TFIIA etc.,- basal transcription .it has number of factors-TFIIA, B,D, E, F,
H.
• TFIID- complex protein consists of TBP and TAFS – 700 KD protein covers a 35 bp region of DNA.
• TBP – 20-40 KD protein binds to 10 bp segment of DNA over TATA box – basal transcription.
• TFIID- support enhanced and basal transcription.
• 2nd class – co activators .ex: TAFs – enhanced transcription co activators.
• Different combination of TAFs with TBP – bind to different promoters and activate them with different
strengths.
• TAFs- basal promoters- initiation complex interacts upstream activators- basal promoters-cell type
specificity, enhance transcription rate convert basal level transcription – enhance the level of activators.
• Sequence element – increase or decrease thee rate of transcription. initiation are enhancer or
repressors.
• Enhancers supress the transcription-silencers or repressors.
• Enhancers or silencers are same in structure except function. enhancers are DNA sequence present
hundreds of bp away from start of gene and enhance transcription stimulate transcription from
promoters.
INITIATION:
• Formation of basal transcription complex.
• Recognition of TATA box by TFIID factor.
• TBP makes sequence- specific contacts with DNA at TATA box through minor groove.
• Most transcription factors bind to major groove.
• Role of TAFs –form an assembly that directs the formed pre-initiation complex to promoters.
• 1. TFIIA- Ist blocks binding of DRI inhibits TFIID activity.
• TFIIB binds and act as a bridge protein for TFIIF – carries RNA polymerase II into complex.
• TFIIB and F – together promote specific interaction between RNA polymerase II and the start site.
• Enzyme facilitate binding of TFIIE – allow bind of TFIIH and J.
• H- important pre initiation complex due to helicase activity controls promoters melting and kinase activity. phoslates the CTD of RNA
polymerase.
• Addition of TFIIE and M final assembly of pre initiation complex or basal transcription complex.
• Binding event extends the size of complex, so 60 bp.
• Binding of TFIIH- release the enzyme from the IC and facilitates promoter clearance leaving IC at the
promoter like bacterial RNA polymerase. Eukaryotic RNA polymerase abortive initiation before
promoter clearance. Ist nucleotide- purine modified by mRNA guanyl transferase to generate a cap.
ELONGATION:

• Encounter secondary structures lead to arrest or termination of transcription .

• To prevent, various elongation factors are associate with RNA polymerase during elongation site side by
side.
• Elongation is slower when compared to bacteria.
• Transcription is longer in the presence of long gene.
• Ex: human dystrophin gene- largest gene- transcription of 2.5 mega base pairs takes 16 hours to
complete.
TERMINATION:
• Poorly characterised.
• RNA polymerase II transcription are processed by polyadenyl at 3’ OH end. Termination is not clear.
• Mechanism of transcription by RNA polymerase by RNA polymerase I and III:
• Transcription of rRNA genes by RNA polymerase I and 5S rRNA , tRNA genes by RNA polymerase II is similarly to
that of RNA polymerase II with minor difference in transcription factor initiate pre initiation complex formation .
• Elongation is same. But termination is different from prokaryotic termination and eukaryotic RNA polymerase II
transcription termination.
• Termination of RNA polymerase II transcription occurs at a site 15 bp to the end of matured RNA and involves
recognition of a specific cis- acting element.
• Termination of RNA polymerase III transcription occurs at sites similarly to bacterial rho- independent
terminators.
POST- TRANSCRIPTIONAL MODIFICATION:

• Transcription and translation not occur simultaneously, occur at 2 different sites.

• Eukaryotic transcription result in primary transcription.
• 3 major changes occur in the primary transcription before it is transport into cytoplasm for translation.
• 1. cleavage of introns.
• 2. addition of t-methyl guanosine gps or mRNA capping.
• 3. poly[A] tail.
MRNA CAPPING

• 1st process.
• Cap- 6 ethyl guanosine attach backward through tri phosphate linkage to 5 terminal end of mRNA .
• The nuclear enzyme –guanyl transferase or mRNA guanyl transferase catalyses addition of guanosine
triphosphate part of cap. This type of cap is called 0 capping.
• Associate with RNA polymerase IC guanine 7- methyl transferase present in cytosol methylates this
guanine.
• Methyl group comes from s- adenosyl methionine and methyl group at 7 position of guanosine.
• In higher eukaryotes methyl group transfer the position of o2 of ribose sugar in next nucleotide. i.efirst
nucleotide in transcription corresponds to position to generate a type 1 cap ex: human.
• 2 methyl groups present one on the guanosine and other is sugar of the other next or +1 residue.
• +1 after guanosine is adenine methyl group – N6 position of adenine o2 of sugar.
• Type 2 cap.
• Triphosphate does not link to 3’ with 5’ .instead the 2 end nucleotide link through 5 sites. Capping of
nucleotide back in to join the growing RNA chain.
• Capping occur before transcription so it is called as co transcriptional process.
• 2 functions:
• 1. it protect mRNA degrade by RNAse
• 2. important to bind mRNA to ribosomes
POLYADENYLATION:

• Most eukaryotic mRNA chain of 40-200 adenine nucleotides attach to 3’ end .poly [A] tail is at transcribed from DNA rather
add after transcription by nuclear enzyme poly [A] polymerase.
• Tails stabilize mRNA and facilitate their exit from nucleus after mRNA enters cytosol, poly [A] tail is shortened.
• Multi subunit complex – trimeric cleavage factors – cleavage and polyadenylation carry out cleavage reactions. enzyme
polyadenylation polymerase[PAP] catalyses the addition of adenylate residue.
• Polyadenylate binding protein attaches to tail and increases the processivity of PAP. Controls the maximum length of
polyadenylate tail.
• Cleavage of polyadenylation complex:
• 2 enzymatic activities: endonuclease and poly[A] polymerase.
• Poly [A] site- with the signal AAUAAA and downstream GU/U region . A complex of CPSF , CFI and II and CS+F bind to these
sequence.
• Cleavage occur and CPSF remains and joined by PAP- synthesis of poly[A] result in addition of 1ST IOAs.
• PAB II – join the reaction stimulate the synthesis of poly[A] extend the tail-200 A residue
• 2 functions of poly[A] mRNA protection and translatability.
• Function of poly[ A] :
• Cap increases the translation 300 fold poly[a] increases the translatability of message by about 20 fold.
• RNA splicing:
• Removal of introns [non coding elements present in RNA molecule]
• Higher eukaryotes- non coding introns separate coding sequences or exons.
• Primary transcription: entire sequence of gene and non coding sequence are spliced out during
processing.
• Splicing mechanism join exon sequence to signal nucleotide codons in exon distal to introns- correct
reading frames.
• 3 introns excision from RNA transcripts.
• Splicing of tRNA precursors:
• excision of introns from yeast tRNA precursors in 2 stage.
• 1st- nm bound endonuclease makes 2 cuts at end of introns. Spicing ligase joint the 2 halves of tRNA to
produce neutral form of tRNA molecule.
• Intron removed- multiple steps.
• 2nd- covalent modification of substrate RNA is cleaved at precise 5 site- ligation.
AUTOCATALYTIC SPLICING OF
TETRAHYMENA:

• Autocatalytic excision of intron in the tetrahymena are RNA precursors no external energy and no
proteins
• Series of phosphodiester bond transfer no bond lost or gained in process.
• The reaction require a guanine nucleotide or nucleoside with a free 3 OH group as a co factor +
monovalent and divalent cations requirement G-3 OH is compulsory.
SPLICING WITH THE HELP OF SPLICEOSOME:

• Introns are removed from primary transcriptase in nucleus . exons are ligated to form mRNA molecule-
spliceosomes . primary transcript -5 small nuclear RNAs nucleoprotein [SnRNp] complex. RNA segment
for the necessary splicing reaction.
RNA EDITING:
• Changes in RNA nucleotide sequence – RNA sequence differs from DNA tem transcribed.
• RNA editing C-U changes, addition of G or C residues and conversion of U-AMG edits of tRNA and
rRNA extremely rare.
• Mid 1980 in mt genes of Trypanosoma and leishmania.
• RNA editing mechanism involves addition and deletion of U/C.addition of upto 560 and deletion of up
to 41 uridine residue.
• Addition or deletion – small RNA molecule of 40 bp length – guide RNA.
• RNA – insertion and deletion at tail region.
• Self splicing of introns – RNA splicing –trans esterification helps of guide RNA- aligns by bp itself with
the unedited RNA. Splits it into 2 and make new bond between broken at tip of tail of gRNA at 3 end.
• RNA editing – post transcriptional process – radical changes in the AA specified by coding.
• 1 step: 2 nucleotide of coding sequences. RNA editing – plant mitochondria , chloroplast and
mammalian nucleus.
• 2 steps: specificity and biochemical modification.
• 3 types:
• Simple editing: conversion of single residue ex: C-U.
• Insertional editing: insertion of single nucleotides or small runs of nucleotides. Transcriptional strand
slipping: ex: G insertion.
• Pan editing: insertion or deleting of multiple cytidine / uridine residues- help of external antisense guide
RNA.
https://www.youtube.com/watch?v=2BwWavExcFI
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