Introduction to DNA Transcription
Synthesis of RNA from DNA template
- The sequence of ribonucleotides in RNA molecule is complementary to the DNTPs in one strand of DNA called the template strand or sense strand.
- The other strand is called the coding strand or anti sense strand because it is identical to the RNA except U for T.
Direction & enzyme involved
- The information in the template strand is read in 3’ -> 5’ direction & the RNA is synthesized in 5’ -> 3’ direction.
- Enzyme involved ->DNA dependent RNA polymerase. The enzyme attaches itself at a specific site on the DNA, the promoter site on the template strand. This is followed by initiation of RNA synthesis
The primary transcript
- hnRNA generated by RNAP is promptly capped by 7-methyl guanosine tri phosphate, which will eventually appear in the mRNA. hnRNA contains both introns and exons.
- The cap is necessary for protection of mRNA from the action of 5’ exonuclease, also for recognition of mRNA by the ribosome
DNA dependent RNA polymerase
- In E coli it exists as a core molecule having 5 subunits. 2 α, ββ‘ w. The core RNAP utilizes a protein factor called sigma factor.
- Core enzyme + sigma factor = Holo enzyme
Steps of RNA synthesis
- Initiation -> binding of holoenzyme to the template at the promoter site to form the initiation complex. This is the closed complex. By sigma factor there is unwinding of 2 DNA strands -> open complex.
- Binding is followed by a conformational change of the RNAP
Initiation Steps continued
- First nucleotide usually a purine associates with the β subunit of the enzyme. This becomes the 5’ of the mRNA.
- In the presence of four RNTPs the RNAP moves to the 2nd base in the template. Now 10-20 nucleotides are polymerized.
- Sigma factor is released
- The enzyme polymerizes the ribonucleotides in a specific sequence that is dictated by template strand and interpreted by Watson and Crick’s base pairing rules. PPi is released at every step.
- A purine ribonucleotides is usually first polymerized into the RNA molecule.
- Unlike DNAP, RNAP does not require a primer and does not have proof reading activity.
- RNAP has unwindase activity which causes local unwinding of the DNA double helix.
- As the RNAP pushes its way between the strands it creates a +ve super coiling ahead and -ve super coiling behind it. Which are released by DNA gyrases and topoisomerases
- Two types -> ‘ rho’ dependent and ‘ rho’ independent.
- ‘rho’ dependent -> here termination is signaled by a sequence in the template strand of the DNA molecule (CA rich region) called ‘rut’ (rho utilization) a signal that is recognized by rho factor.
- Rho protein has an ATP dependent helicase activity.
Rho independent termination
- The RNA transcript must be able to form a stable hairpin turn that slows down the progress of RNAP. The hairpin turn is complementary to a palindrome sequence
- Following the hairpin turn the RNA transcript must have a string of ‘U’ s. the A=U bonding is weak which facilitates its separation from the DNA strand.
- After termination the core enzyme separates from the DNA template.
- With the assistance of another sigma factor the core enzyme recognizes another promoter at which the synthesis of a new RNA molecule commences
Transcription of eukaryotic gene
- Far more complicated process than that in prokaryotic gene.
- In addition to RNAP recognizing the promoter region and initiating RNA synthesis , a number of transcription factors bind to distinct sites of DNA.
- There are 3 classes of RNAP in the nucleus.
RNAP – classes
- RNAP1 -> synthesizes the 45S precursor of r RNA (5.8s, 18s, 28s)
- RNAPII -> synthesizes the precursor of mRNA and some snRNAs.
- RNAPIII -> synthesizes smaller RNAs – 5srRNA, tRNA, snRNA.
Post transcriptional modification
- A primary transcript is a linear copy of a transcriptional unit, the segment of DNA between initiator and terminator sequences.
- The primary transcript of both tRNAs and rRNAs are post transcriptionally modified by cleavage of the original transcripts by ribonucleases. tRNAs are further modified.
r RNAs and t RNAs
- In eukaryotes single 45 s precursor gives rise to 5.8s, 18s and 28s r RNAs. 5s rRNA is produced from a separate precursor molecule.
- 30 s precursor in prokaryotes produces 5s 16 s and 23 s r RNAs.
- Both pro and eukaryotic t RNAs are made from longer precursors that must be trimmed.
- Prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic mRNA is extensively modified after transcription.
- tRNAs and rRNAs of both pro and eukaryotes are modified after transcription.
Post transcriptional modification of mRNA
- Addition of poly A tail
- Removal of introns
- RNA editing
- The cap is 7 methyl guanosine tri phosphate linked to the 5’ terminal of the mRNA. The addition is catalyzed by the enzyme guanylyl transferase.
- The methylation of the terminal guanine is catalyzed by the enzyme 7 methyl transferase. S adenosyl methionine is the methyl donor.
Function of the CAP
- Facilitates the initiation of translation.
- Helps the recognition of mRNA by the protein synthesizing machinery (ribosome)
- Protects the mRNA from the action of exonuclease
Addition of poly A tail
- A chain of 40 – 200 adenine nucleotides is attached to the 3’ end of the primary transcript. This is added by the enzyme poly A polymerase.
- Most eukaryotic mRNAs have poly A tail except histone mRNA.
- Function is to stabilize the mRNA. After the mRNA enters the cytosol the poly A tail is gradually shortened.
- From the primary transcript introns are removed and the exons are spliced together to form mature mRNA.
- snRNAs are associated with proteins to form snRNPs (small nuclear ribonucleo proteins ) which facilitates removal of introns and splicing of exons.
- After intron removal mature mRNA leave the nucleus and enter the cytosol.
- Central dogma is DNA -> RNA -> Protein. So change in DNA will be reflected into RNA and into protein, but sometimes the coding information can be changed at the RNA level. This is called RNA editing.
- example -> apo B 100 and apo B 48, both are synthesized from the same mRNA by RNA editing.
- SNRNP, associated with hnRNA at the exon- intron junction form spliceosome. It consists of hnRNA, 5 snRNAs ( U1, U2, U4, U5, U6) and more than 50 proteins. collectively they are also called ‘SNURP’.
- SNURPs position the RNA segments for splicing, cut the exon- intron junction, joins the exons to form a continuous sequence after intron removal.
- They are RNA molecules with catalytic activity. Several enzymatic actions have been attributed to RNA.
- SnRNA -> intron removal and splicing.
- Peptidyl transferase -> protein synthesis.
- RNAse P -> modification of tRNA precursor.
- They have specificity and obey M.M. kinetics.
Inhibition of RNA Polymerase
- Actinomycin -D -> Elongation of RNA chain is inhibited both in pro and eukaryotes.
- Rifampicin -> inhibits bacterial RNAP.
- Acridine -> acts in the same way as actinomycin – D.
- 3’ deoxy adenosine -> causes premature chain termination.
- α- aminitine -> inhibits RNAPIII.
Promoter Region (prokaryote)
- The promoter sequences are clustered approximately 10 base pairs and 35 base pairs upstream (-10 and – 35 sequences) from the transcription start site.
- 5‘TATAAT3’ for the -10 bp sequence ( Prinbow box) and 5’TTGACA’ for – 35 sequence.
- All promoter sequences are recognized by the same sigma subunit of the RNAP.
- -25 to -30 bp upstream from the transcription start site 5’TATAAAAG 3’ (TATA or hogness box).
- Sequence further upstream G.C box and CAAT box.
- Enhancer and repressor sequence, both upstream and downstream.
INHIBITOR OF TRANSCRIPTION
- Rifampicin inhibits RNAP in bacteria.
- Actinomycin D inhibits RNAP in bacteria and eukaryotes ( wilms tumour)
- α Amanitin inhibits eukaryotic transcription.
- Synthesis of RNA from DNA is known as Transcription.
- The transcribable portion of DNA is called Gene.
- During transcription one strand of DNA acts as the template strand and the other as the coding strand.
- RNA synthesis is catalysed by the enzyme RNA polymerase.
- RNAP recognises the promoter element on the template strand as the transcription start site.
- A single molecule of RNAP catalyses the synthesis of all types of RNA in prokaryotes.
- 3 distinct RNAPs catalyse the transcription in eukaryotes.
- Post transcriptional modification occurs in eukaryotes.
- RNA acting as an enzyme is called a Ribozyme.
- Reverse transcriptase synthesizes complimentary DNA from viral RNA in retroviruses.
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