Introduction to Transcription:
Transcription is the biological process in which an RNA strand is generated based on a DNA template, with the reaction being facilitated by the enzyme RNA polymerase.
Similarities Between Transcription and Replication:
- Template for both the processes is DNA.
- The direction of synthesis of both DNA and RNA is 5′-3′
- Both processes entail the polymerization of nucleotides, where the individual nucleotides are linked together through the formation of phosphodiester bonds.
- Both processes involve the breakage of parental double helix.
- Both the processes broadly accomplish through the initiation, elongation and termination.
Differences between transcription and replication:
| Characteristics | Replication | Transcription |
| Definition | DNA replication is the biological process of duplication of the double helical structure of DNA to produce two identical daughter molecules each containing two complementary strands antiparallel in orientation. | Transcription is the process of synthesis of complementary strand of RNA using template DNA, catalyzed by RNA Polymerase enzyme. |
| Template | Double stranded DNA | Single stranded DNA(asymmetric |
| Raw material for addition | dNTPs | rNTPs |
| Product | Double Stranded DNA | Single stranded RNA |
| Enzyme catalyzing polymerization | DNA Polymerase | RNA Polymerase |
| Need for primer | Yes( No de novo synthesis) | No( denovo synthesis) |
| Bases involved ; sugar involved | Adenine, Guanine, Cytosine, Thymine ; Deoxyribosugar | Adenine, Guanine, Cytosine, Uracil ; Ribosugar |
| Processing | No post DNA replication modifications reported | Post transcriptional modification is vital in case of eukaryotes |
Structure of RNA polymerase (RNAP):
The best studied RNA polymerase is from E. coli hence is chosen as a model organism. A single RNA polymerase is believed to be responsible for synthesis of mRNA, rRNA, tRNA. The holoenzyme is approximately 460KD. It recognizes the promoter region on the DNA that is to be transcribed. It next makes a complementary RNA copy of the DNA template strand. It comprises of:
Core enzyme
Core enzyme is made up of four peptides, 2α, 1β, and 1β’. 2α aids enzyme assembly Template binding requires 1β, and 5’→3′ RNA polymerase activity requires 1β.
Holoenzyme
RNA polymerase can identify promoter areas on DNA thanks to the σ subunit. The σ subunit together with core enzyme forms holoenzyme.
Active site and cofactor
The shape of each enzyme is similar to that of a crab’s claw, with the two gripping extensions made up mostly of the biggest subunits of the enzyme, β and β’. The catalytic core, or active site, is located in the “active center cleft,” which is tucked away at the base of these claw-like extensions. This active site functions by the well accepted two-metal ion mechanism that all polymerases employ during nucleotide inclusion. It is composed of components from both main subunits. Interestingly, one Mg²⁺ ion stays securely in the active site in this particular system, but the second enters bound to the incoming nucleotide and leaves with the pyrophosphate after the addition event is over.
Fig: Structure of RNA polymerase (RNAP)
| Subunit | Molecular Weight (kD) | Gene | Function |
| 2α | 40 each | rpoA | enzyme assembly and determination of DNA to be transcribed |
| β | 155 | rpoB | catalyse polymerization |
| β’ | 160 | rpoC | bind and open DNA complex |
| σ | 70 | rpoD | Promoter recognition |
Mechanisms of Transcription Termination:
The transcription elongation halts when termination signal is reached, facilitated by the action of the translocator TRCF. This termination is triggered by damaged DNA or by other unanticipated hindrances. But termination is a normal and important function whereby elongating polymerase dissociate from the DNA and release the RNA chain it has made. In bacteria, terminators come in two types: a. ρ-dependent termination b. ρ-Independent termination.
Rho (ρ) -Dependent termination
ρ-Dependent termination needs a protein termed, rho (ρ) protein for ceasing the process of transcription. The ring-shaped Rho protein attaches itself to the C-rich “rho recognition site” close to the 3′ end of the freshly created RNA. ATPase and helicase activities are also present in Rho protein. The rho protein travels along the freshly generated RNA by means of its ATPase activity until it reaches the RNA polymerase that has stalled at the termination point.
The ATP dependent helicase activity of ρ separates the RNA-DNA hybrid helix, causing the release of the RNA and RNA polymerase and rho protein also fall off.
Fig: Rho-dependent transcription termination
Rho (ρ)-Independent termination
For who-independed termination of transcription, a sequence in the DNA template has to generate a sequence in the newly formed RNA that is self-complementary. GC rich stem plus a loop formed as a result of folding back of the RNA. Structure resembling “hairpin” forms. This causes the RNA polymerase to stop. The RNA transcript also has a string of U at the 3′-end, just past the hairpin. These U base pairs form an extremely unstable connection with the A base pairs of the DNA. It is also called Intrinsic termination, since no rho-protein is required.
Fig: Rho-independent transcription termination
Role of Nus Factors in Transcription Termination
Also, Nus factors are involved that increase the efficiency of termination of transcription. Nus factors are proteins involved to increase the efficiency of termination of transcription. Nus A is a general transcription factor that elevates the efficiency of termination by enhancing the tendency of RNA Polymerase to pause at terminators. Nus B and S10 form dimer that bind specifically to RNA containing Box A. Nus G is thought to be concerned with general assembly of Nus Factors with RNA Polymerase.