PROCESS OF TRANSCRIPTION:-

The process of transcription involves following steps:-

A) Activation of Ribonucleotides.

The ribonucleotides occur freely in the nucleoplasm. Before transcription starts, the nucleotides are activated via phosphorylation for which the enzyme phosphorylase and energy are required. The activated ribonucleotides are adenosin

e triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP) and cytidine triphosphate (CTP).
B) DNA Template.

On specific signals, segments of DNA corresponding to one or more cistrons become derepressed and ready to transcribe.

Enzymes required for chain separation are unwindases and helix destabilizing proteins or gyrases. Terminator region has either poly a base sequence or palindromic sequence.
C) Base Pairing.

Ribonucleoside triphosphates present in the surrounding medium come to lie opposite the nitrogen bases of the DNA template strand. They form complementary pairs and then the two extra phosphates present in the ribonucleoside triphosphates (ribonucleotide diphosphates) separate with the help of pyrophosphatase. Energy is released in the process.
D) Chain Formation. With the help of RNA polymerase the adjacent ribonucleotides held over DNA template join to form RNA chain. Once the RNA chain is initiated the sigma (s) factor separates. RNA polymerase core enzyme now moves along the DNA template elongating the chain at the rate of some 30 nucleotides per second. There is no proof reading during transcription. RNA synthesis stops as soon as polymerase reaches the terminator region. Rho factor (p) is required for this. Terminator region has a stop signal. It also possesses 4 - 8 A-nucleotides.
E) Separation of RNA.

Termination or rho factor has ATP-ase activity. It helps in the release of the completed RNA chain which is called a primary transcript. It undergoes processing to form functional RNAs. In many prokaryotes, the structural genes of related functions are grouped together in operons. An operon is transcribed as a single unit resulting into a polycistronic mRNA. In eukaryotes, transcription unit is monocistronic.
F) Duplex Formation.

After the release of primary transcript, the two strands of DNA again establish linkages between the complementary base pairs. The enzymes Gyrases, unwindases and HD proteins are released. Finally the double helical form of DNA is resumed.
G) Post-Transcription Processing.

Primary transcript is larger than the functional RNAs and is called heterogeneous or hnRNA (especially in case of mRNA). Primary transcript is converted into functional RNAs by these post transcription processes. They are of four types:
(i) Cleavage

Larger RNA precursors are cleaved to form smaller RNAs. Primary transcript of rRNA in eukaryotes is 45S. Primary transcript forms 5-7 tRNA precursors on being cleaved by ribonuclease-P (an RNA enzyme).

(ii) Splicing.

Eukaryotic transcripts possess extra segments (introns or intervening sequences). snRNAs along with some protein molecules function as 'enzymes for splicing. They are called SnRNPs or small nuclear ribonucleoproteins . SnRNPs get attached to 5' and 3' ends on introns. With the help of some more proteins, a complex called spliceosome develops. It requires energy from ATP Spliceosome removes the intron and joins the exons to produce mature mRNA. A ligase may be required for this. Ribozyme (an-RNA enzyme) is a self-splicing intron involved in some of these reactions as well as catalyzing polymerization. Self-splicing is usually seen in some primary r-RNA transcripts. The split-gene (genes having introns) arrangements represent probably an ancient feature of the genome. The presence of introns is reminiscent of antiquity, and the process of splicing represents the dominance of RNA-world.
(iii) Terminal Additions.

Some extra nucleotides are added to the ends of RNAs and they serve specific functions, E.g., CCA segment in tRNA, cap nucleotides at 5' end of mRNA or poly-A segments at 3' end of mRNA. Addition of cap nucleotides at 5' end is called capping. mRNA cap is formed from GTP & is called 7 methylguanosine (7MG). It is required for ribosomal recognition.
(iv) Nucleotide Modifications.

Certain nucleotides are methylated, ethylated, deaminated etc. to produce different chemicals. These are most common in tRNA- methylation (E.g., methyl cytosine, methyl guanosine), deamination (E.g., inosine from adenine), dihydrouracil, pseudouracil, etc.

In bacteria, since the mRNA does not require any processing to become active, and also since transcription and translation takes place in the same compartment (there is no separation of cytosol and nucleus in bacteria), many times the translation can begin much before the mRNA is fully transcribed. Consequently, the transcription and translation can be coupled in bacteria.

In vitro synthesis of RNA was first performed by Ochoa (1967). He discovered polynucleotide phosphorylase which could polymerize ribonucleotides to produce RNA without any template.