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5,6.RNA TRANSCRIPTION

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9 views49 pages

5,6.RNA TRANSCRIPTION

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Reshma RB
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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RNA

• RNA : polymer of purine and pyrimidine ribonucleotides linked


together by 3′,5′-phosphodiester bonds.
RNA Distribution:
• Ribosomes & Endoplasmic reticulum - 50%
• Cytoplasm - 25%
• Mitochondria - 15%
• Nucleus - 10%
MainTypes of RNA
• Messenger RNA (mRNA)
• Transfer RNA (tRNA)
• Ribosomal RNA (rRNA)
mRNA
• Class that is most heterogeneous in abundance, size and stability.
• Function as messengers conveying the information in a gene to the
protein-synthesizing machinery
• mRNA serves as a template on which a specific sequence of amino
acids is polymerized to form a specific protein molecule.
• 2% to 5% of total eukaryotic cellular RNA.
• Transfer RNA (tRNA) (Soluble RNA)
• Serve as adapters for the translation of the information in the
sequence of nucleotides of the mRNA into specific amino acids.
• Vary in length from 74 to 95 nucleotides.
• Atleast 20 species of tRNA molecules are present in every cell.
• All tRNA molecules allows extensive folding and intrastrand
complementarity to generate a secondary structure that appears
in two dimensions like a cloverleaf.
Ribosomal RNA (rRNA)
• Ribosome is a cytoplasmic nucleoprotein structure that acts as the
machinery for the synthesis of proteins from the mRNA templates.
• Components of mammalian ribosomes:
Prokaryotic ribosome
Components of Mammalian Ribosomes

RNA
TRANSCRIPTION
DEFINITION AND SALIENT FEATURES
• The process by which RNA is synthesised from DNA is known as
transcription.

CENTRAL DOGMA OF MOLECULAR BIOLOGY


• Only one of the strand of DNA is copied; genes may be on any strand.
• Only certain areas of DNA is copied .
• Base pairing rule obeyed; T replaced by U
• Ribonucleotides are added.
• Transcription occurs in the 5’ to 3’ direction.
• No RNA primer needed.
• No efficient proof reading mechanism.
• The template strand is read in 3’ to 5’ direction.
• Strand of DNA transcribed to RNA is called Template Strand / Non-
coding or antisense strand.
• Opposite strand is called Anti template strand / Coding or sense
strand.
• mRNA is complementary to the DNA strand that is copied.
• Transcription is accomplished by DNA dependent RNAP.
• In Eukaryotes:
Type Function Sensitivity to α
Amanitin

RNAP I or A Synthesis of rRNA Not inhibited

RNAP II or B Synthesis of mRNA Inhibited

RNAP III or C Synthesis of tRNA Moderately sensitive


• In Prokaryotes:
• Only one type of RNAP
• 2 identical Alpha units
• 2 Beta subunits –not identical (β & β’)
• One ω subunit α α

σ
β’
β
• Transcription unit
• Region of DNA that includes the signals for initiation, elongation
and termination of transcription : promoter area, the gene to be
transcribed and terminator site.
• Promoters are specific areas of DNA which act as starting signals for
initiation of transcription.
• Located upstream or downstream from start site.
• AT rich areas
• RNAP attaches to the promoter area and initiates transcription.
By convention, the promoter DNA regulatory sequence elements such as the -35 and the -10 TATA
elements are described in the 5′→3′ direction and as being on the coding strand.
• INITIATION OF TRANSCRIPTION
• Signals for initiation of transcription
• In prokaryotes
• 35 bp upstream from TSS : 5’TGTTGACA3’
• 10 bp upstream : 5’TATAAT3’(TATA Box or Pribnow Box)
• In eukaryotes
• About 25-30 bp upstream from TSS : 5’TATAAA (Goldberg-
Hogness Box)
• 70 to 80 bp upstream: CAAT Box( GGCCAATCT)
Coding strand GGCAATCT TATAAA
5’ -80 bp - 25bp

3’
Transcriptiion
Template strand initiation site
• Initiation
• In Bacteria
• The starting point of transcription corresponds to the 5'
nucleotide of the DNA, designated as +1.
• The DNA helix partially unwinds and RNAP binds with the
promoter site on DNA with the help of sigma factor forming
pre-initiation complex (PIC).
• When it reaches the appropriate site on the gene, the first
nucleotide of the mRNA attaches to the initiation site on the beta
subunit of RNAP. This becomes the 5' end of the mRNA. It will be
complementary to the base present in the DNA at that site.
• The next nucleotide attaches to the RNAP. A phosphodiester bond
is formed. Then the enzyme moves to the next base on the
template DNA.
• After RNA chain length reaches ~10–20 nt, the polymerase
undergoes a conformational change,σ factor is released and move
away from the promoter, transcribing down the transcription unit:
Promoter Clearance.

• In Eukaryotes:
• Transcription factors are involved(TF II).
• TATA box recognised by TATA binding protein (TBP).
• SL1 factor ensures that RNAP could locate the start point
Eukaryotic basal transcription complex
ELONGATION OF TRANSCRIPTION
• RNAP moves along the DNA template.
• New nucleotides are added to nascent RNA one by one according
to the base pairing rule.
• As the RNAP moves on the template strand, DNA helix unwinds
down stream and winds upstream and produces a transcription
bubble - containing RNAP, DNA and nascent RNA (~20 bp).
• TERMINATION OF TRANSCRIPTION
• In prokaryotes
• Rho (ρ ) Dependent Termination
• ATP required.
• Rho factor attaches to specific signals in DNA .
• RNAP cannot move.
• Nascent RNA released.
• Rho independent termination
• GC rich sequences which contain interrupted inverted repeat
sequences precedes the sequence of 6-7 U residues in the RNA
chain.
• A stem and loop structure is formed which facilitates
dissociation of primary transcript from DNA.
• In eukaryotes:
• Poorly understood.
• Termination signals exist far downstream of the coding
sequence of genes.
• Post- transcriptional processing of mRNA

• RNA formed and released from template DNA : heteronuclear mRNA or


primary transcript.
• Extensive modifications of hnRNA occurs in the nucleus forming mature
mRNA.
• 5’ Capping
• Addition of Poly A tail
• Methylation
• Removal of introns
• Joining of exons - splicing
• 5’ capping
• All eukaryotic mRNAs are capped at the 5’ end by 7-methyl
Guanosine tri phosphate residue in the nucleus.
• For identifying the specific mRNA by the translating machinery.
• Protection of the 5′ end of mRNA from attack by 5′ → 3′
exonucleases.
• Poly A tailing

• Poly(A) polymerase adds a poly(A) tail which is subsequently


extended to as many as 200 A residues.
• Protects the 3′ end of mRNA from 3′ → 5′ exonuclease attack and
facilitates translation.
• Methylations
• N6 of Adenine
• 2’ OH group of ribose.
• Removal of introns & splicing of exons
• Coding regions (exons) are joined together and non coding
regions(introns) are cleaved in the nucleus by spliceosomes.
Untranslated regions of mRNA
• Even after the processing, mature mRNA is 2 or 3 times larger than
the size required for coding of proteins (non coding sequences).
• This is because long stretches of untranslated regions(UTR) are
present both at 3’ and 5’ ends.
• The exact function of 5’ UTR and 3’ UTR sequences is unknown, but
they have been implicated in RNA processing, transport, degradation
& translation
Inhibitors of RNA Synthesis
Inhibitors of RNA synthesis

Inhibitor Source Mode of action


Actinomycin-D Antibiotics from Insertion of phenoxazone ring
streptomyces between two G-C bp of DNA

Rifampicin Synthetic derivative Binds to beta subunit of RNA


of rifamycin polymerase which is inactivated

Alpha Toxin from Inactivates RNA polymerase II


amanitin mushroom
3'–deoxy Synthetic analog Incorrect entry into chain causing
adenosine chain termination
Reverse transcriptase
• Genetic materials of some animal and plant viruses are made up of
RNA. Eg: HIV,SARS CoV-2
• RNA acts as a template on which a single strand DNA is synthesized by
RNA dependent DNAP (Reverse transcriptase).
• RNA strand is removed from RNA-DNA hybrid by RNAse H.
• Another strand of DNA is synthesized by DNA Polymerase.
• A process against central dogma.
• This double stranded viral DNA is then integrated into the host cell
DNA.
• The viral genes are transcribed and translated by the host cell
mechanisms.
• Some tumor viruses also possess reverse transcriptase activity .

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