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DNA TRANSCRIPTION
Rikesh Lal Shrestha
M.Pharm (2015), Industrial
Kathmandu University
Contents• INTRODUCTION
• DNA TRANSCRIPTION STAGES
• POST TRANSCRIPTION MODIFICATION
5’ CAPPING
POLY (A) TAIL
SPLICING
• REVERSE TRANSCRIPTION
• DRUGS INHIBITING TRANSCRIPTION
Introduction• The synthesis of an RNA molecule from DNA molecule in the
presence of enzyme RNA polymerase is called Transcription.
• Information stored in DNA is transferred to RNA moleculesduring transcription.
• All eukaryotic cells have five major classes of RNA:ribosomal RNA (rRNA), messenger RNA (mRNA), transferRNA (tRNA), small nuclear RNA and microRNA (snRNA andmiRNA).
• The first three are involved in protein synthesis, while thesmall RNAs are involved in mRNA splicing and regulation ofgene expression.
Similarities between Replication and Transcription
The processes of DNA and RNAsynthesis are similar in thatthey involve-
(1) The general steps ofinitiation, elongation, andtermination with 5' to 3'polarity;
(2) Large, multicomponentinitiation complexes; and
(3) Adherence to Watson-Crickbase-pairing rules.
Replication Transcription
DNA replication is catalyzed by DNA
polymerase which needs a primer.
RNA synthesis is catalyzed by RNA
polymerase
Deoxyribonucleotides are used in DNA
synthesis
Ribonucleotides are used in RNA
synthesis
Thiamine is the complementary base pair
for Adenine in DNA.
Uracil is the complementary base pair for
Adenine in RNA.
The entire genome must be copied during
DNA replication
Only a portion of the genome is
transcribed or copied into RNA
There is proofreading function during
DNA replication.
There is no proofreading function during
RNA transcription.
Differences between Replication and Transcription
Template strand•The DNA strand that is transcribed or copied into an RNA molecule is called template strand.
•The another non-template DNA strand, is called coding strand.
•The information in the template strand is read out in the 3' to 5' direction
•The information in the RNA molecule is read out in the 5' to 3' direction
•In the coding strand the sequence is same as that of the sequence of RNA primary transcript. With the exception of T for U changes.
Transcription unit• A transcription unit is defined as that region of DNA that includes the signals for
transcription initiation, elongation, and termination.
• The nucleotide in the promoter adjacent to the transcription initiation site is designated -1,
• These negative numbers increase as the sequence proceeds upstream, away from the
initiation site
• The nucleotide in the initiation site is designated +1,
• These positive numbers increase as the sequence proceeds downstream, away from the
initiation site
Bacterial DNA-Dependent RNA Polymerase
The DNA-dependent RNA polymerase (RNAP) of the bacterium Escherichia coli exists as an approximately 400 kDa core complex consisting of-
•two identical α subunits,
•similar but not identical β and β ' subunits, and
•an ω subunit and a
•A sigma subunit (σ)
•Beta is thought to be the catalytic subunit.
Bacterial DNA-Dependent RNA Polymerase
• RNAP, a metalloenzyme, also contains two zinc molecules.
• The core RNA polymerase associates with a specific protein factor (the sigma σ factor) that helps the core enzyme recognize and bind to the specific deoxynucleotide sequence of the promoter region to form the preinitiationcomplex (PIC)
• Bacteria contain multiple factors, each of which acts as a regulatory protein.
Mammalian DNA-Dependent RNA
Polymerases
Mammalian cells possess three distinct nuclear
DNA-Dependent RNA Polymerases
• RNA polymerase I is for the synthesis of
rRNA
• RNA polymerase II is for the synthesis of
mRNA and miRNA
• RNA polymerase III is for the synthesis of
tRNA/5S rRNA, snRNA
Prokaryotic versus Eukaryotic TranscriptionParameters Prokaryotic Cell Eukaryotic Cell
Location Transcription Occur in cytoplasm of Prokaryotic cell
Transcription Occur in nucleus of Eukaryotic cell
RNA Polymerase
Single RNA polymerase have capability to produce rRNA, mRNA & tRNA.
Three classes of RNA polymerases (I, II,III). RNA Poly. I rRNA, RNA Poly.II
mRNA, RNA Poly.III tRNA
Synthesized RNA
The RNA produce is fully functional for translation process.
The RNA produced is not fully functional and k/a premature m-RNA which need to be modified to produce mature m-RNA that can synthesize protein.
Process Transcription along with translation process take place. So the process is also k/a transcription coupled translation.
After transcription complete then only translation process take place.
TranscribedRNA
Transcribed RNA is polycistronic (i.e multiple genes are regulated by only one promoter) . So, multiple proteins are produce in chain.
Transcribed RNA is monocistronic (i.esingle gene is placed on single promoter)
Stages of Transcription
Both prokaryote and eukaryote have 3 stage of transcription. They are:
• Initiation
• Elongation
• Termination
Initiation in Prokaryote• The RNA polymerase and sigma factor combine to form a molecule
called holoenzyme.
• This holoenzyme binds to the promoter region of double strands DNAforming closed complex.
• Then holoenzyme unwind DNA helix , thus open promoter complex isformed.
• After 10–20 nucleotides have been polymerized, holoenzyme undergoesconformational change leading to promoter clearance.
• Once this transition occurs, holoenzyme moves away from the promoter,transcribing down the transcription unit, leading to the next phase of theprocess, elongation.
Elongation in Prokaryote• As the elongation complex that is holoenzyme progresses along the DNA
strands, it unwinding DNA to provide access for the complementary basepairing to the nucleotides of the template strand.
• After addition of few riboneucleotides the sigma factor dissociate fromholoenzyme . Then RNA polymerase adds nucleotides to 3’ –OH group.Thus elongation proceed in the direction of core enzyme.
Termination in Prokaryote• Two types of terminator sequences
occur in prokaryotes:
•Rho() Independent: In RNA sequencethere occurs G and C rich region, theirPalindromic repeat forms a hairpin loop.Beyond the hair pin, RNA sequencecontains a strings of Us, the bonding ofUs to the corresponding As is weak. Thisfacilitates the dissociation of the RNAfrom DNA.
•Rho() dependent: A sequence of DNAtemplate strand signal is recognized byprotein k/a Rho () factor proteins, itattach and climb RNA strand then breakthe hydrogen bonds between thetemplate DNA and RNA .
Initiation in EukaryoteWe can divide eukaryotes promoter into two regions:
The core promoters elements. The best characterized are TATA Box = TATAAAA, located at about position -30 bp(*AT-rich DNA is easier to denature than GC-rich DNA)
Promoter proximal elements (located upstream, ~-50 to -200 bp) “Cat Box” = CAAT and “GC Box” GGGCGG
General Transcription factors (GTF) are proteins that help eukaryotic RNA polymerase recognize promoter sequences.
Binding of GTFs and RNA polymerase occurs in set of order.
Complete complex (RNA polymerase + GTFs) is called a pre-initiation complex(PIC).
Transcription regulatory proteins (activators) bind to the enhancers region in ds DNA and also to RNA polymerase II in order to increase the rate of transcription initiation of eukaryotic genes.
Initiation in EukaryoteOrder of binding is: IID + IIA + IIB + RNA poly. II + IIF +IIE +IIH
Elongation in Eukaryote• As RNA polymerase moves along the DNA it continues to
untwist the double helix, exposing about 10 to 20 DNA basesat a time for pairing with RNA nucleotides.
• RNA polymerase synthesizes a single strand of RNA againstthe DNA template strand (anti-sense strand), addingnucleotides to the 3’ end of the RNA chain.
Elongation
RNA
polymerase
Non-template
strand of DNARNA nucleotides
3 end
A E G C A
U
T A G G T T
AT C C A A
3
5
5
Newly made
RNA
Direction of transcription
(“downstream”) Template
strand of DNA
Termination in Eukaryote• Specific sequences in the DNA signal termination of
transcription (AAUAAA)
• When one of these is encountered by the polymerase, theRNA transcript is released from the DNA and the doublehelix can zip up again.
POST TRANSCRIPTION MODIFICATION5’ capping
• It is a 7-methylguanosine tri-phosphate cap structure at the5' terminal of eukaryotic mRNA.
• The cap structure is added to the 5' end of the newlytranscribed mRNA before it transport to cytoplasm.
• The 5' cap of the RNA transcript is required both for efficienttranslation initiation and protection of the 5' end of mRNAfrom attack by 5-'3' exonucleases.
5’ capping•There is addition of the Guanosine triphosphate to 5’terminal of mRNAby enzyme guanylyl transferase.
•The 5’-end of the mRNA is capped 5’ to 5’ with a guanine nucleotideforming triphosphate bridge.
•Methylation occurs in N7 of guanine by guanine-7-methyl transferase.
•Additional methylation steps may occur.
•The secondary methylations of mRNA molecules, those on the 2'-hydroxy and the N6 of base residues, occur after the mRNA molecule hasappeared in the cytoplasm.
Poly (A) tail• Poly(A) tails are added to the 3' end of mRNA molecules in a posttranscriptional
processing step.
• The mRNA is first cleaved about 20 nucleotides downstream from an AAUAAA
recognition sequence.
• Another enzyme, poly(A) polymerase adds a poly(A) tail which is subsequently
extended to as many as 200 A residues.
• The poly(A) tail appears to protect the 3' end of mRNA from 3' -5' exonuclease
attack.
RNA Splicing• Splicing : Removal of introns
• Introns: non-coding sequences
between exons
• Exons: amino acid coding sequences
are spliced.
• “snurps” snRNP bind to splice site and
form a spliceosome
• Spliceosome excises the intron and
rejoins the exons.
• Mature mRNA is produce that moves
into cytosol from nuclear pore for
protein synthesis.
Reverse Transcription• It is the process of synthesis double stranded
DNA from Single stranded RNA by reverse
transcriptase enzyme (RNA directed DNA
polymerase).
• Reverse transcriptase common in HIV,
MMLV(Moloney Murine Leukemia Virus),
AMV(Avian Myeloblastosis Virus)
• Reverse transcriptase enzyme includes two
activity: DNA polymerase and RNAase H
Retrovirus Cycle
Drug Inhibiting Transcription• Rifampicin binds with Beta subunit of
prokaryotic RNA polymerase but not toeukaryotic RNA polymerases. Rifampicin usefor the treatment of tuberculosis and leprosy.
• Mitomycin used as anticancer drug Intercalates with DNA strands blocks transcription.
• Alpha amanitin is a molecule made from the“death cap” mushroom and is a known potentinhibitor RNA polymerase. The mechanism ofaction is that alpha amanitin inhibits RNApolymerase –II at both the initiation andelongation states of transcription.
• Actinomycin D- Intercalates with DNA strands .Actinomycins inhibit both DNA synthesis and RNA synthesis by blocking chain elongation. Actinomycins are used as anticancer drugs
REFERENCE• Professor (Dr.) Namrata Chhabra Biochemistry For Medics-
Lecture Notes
• Chapter 12 of Molecular Biology of the Gene 6th Edition(2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine,M, Losick, R. 377-414.
• Murakami KS, Darst SA. (2003) Bacterial RNA polymerases:the wholo story. Curr Opin Struct Biol 13:31-9.
• Campbell, E, Westblade, L, Darst, S., (2008) Regulation ofbacterial RNA polymerase factor activity: a structuralperspective. Current Opinion in Micro. 11:121-127
• John Wiley & Sons, Inc, Transcription and RNA Processing
• Biochemistry For Medics- Lecture Notes.
THANK YOU……!!!!