Transcription

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

Initiation

  • 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.

Elongation

  • 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

Termination

  • 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.

Termination

  • 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.

Messenger RNA

  • 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

  • Capping
  • Addition of poly A tail
  • Removal of introns
  • Methylation
  • RNA editing

CAPPING

  • 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. 

Intron removal

  • 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.

RNA editing

  • 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.

Spliceosome

  • 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.

Ribozyme

  • 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.

Promoter (eukaryote)

  • -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.

SUMMARY

  1. Synthesis of RNA from DNA is known as Transcription.
  2. The transcribable portion of DNA is called Gene.
  3. During transcription one strand of DNA acts as the template strand and the other as the coding strand.
  4. RNA synthesis is catalysed by the enzyme RNA polymerase.
  5. RNAP recognises the promoter element on the template strand as the transcription start site.
  6. A single molecule of RNAP catalyses the synthesis of all types of RNA in prokaryotes.
  7. 3 distinct RNAPs catalyse the transcription in eukaryotes. 
  8. Post transcriptional modification occurs in eukaryotes.
  9. RNA acting as an enzyme is called a Ribozyme.
  10. Reverse transcriptase synthesizes complimentary DNA from viral RNA in retroviruses.

Other Biochemistry Books

. Biochemistry of Proteins

Carbohydrate Metabolism

VITAMIN PYRIDOXINE AND BIOTIN

Vitamin A

Pyrimidine metabolism

Purine Metabolism

Cardiac Biomarkers

VITAMIN D

. Blood Metabolism (Heme synthesis and breakdown)

DNA Repair

Regulation of gene expression

DNA Replication

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