DNA Transcription: Steps and Mechanism

The synthesis of the single-stranded RNA, complementary to one of the DNA strands is called transcription. The genetic information encoded in the double-stranded DNA is transformed into the RNA strand by the action of the enzyme RNA polymerase. Transcription produces three types of RNA: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). 

• mRNA: carries instruction for protein synthesis
• tRNA: transports the aminoacids
• rRNA: component of the ribosomes 

Among these three types of RNA, only messenger RNA (mRNA) is translated into proteins. 

Double-stranded is made up of two single strands. Each of these strands has its function. One strand is the template strand (non-coding strand, antisense strand), and another is the non-template strand (coding strand, sense strand). The template strand serves as a template for RNA synthesis, i.e., if adenine (A) is encoded in the template strand, uracil (U) will be encoded in the newly synthesized RNA. If guanine (G) is encoded in the template strand, then cytosine (C) will be encoded in the RNA.

Steps in Transcription (RNA synthesis)

Transcription in prokaryotes (e.g., E. coli) is divided into three steps: initiation, elongation, and termination.

Initiation

Transcription begins by binding the RNA polymerase holoenzyme to the promoter region in DNA. The promoter region is the specific sequence in the DNA that is the initiation site for transcription.

RNA polymerase consists of multiple subunits. It consists of core enzyme and holoenzyme. The RNA polymerase helps in unwinding the DNA double helix. Then one of the strands functions as a template.

• Core enzyme consists of 2α, β and β’. The core enzyme lacks specificity and cannot recognize the promoter region of the DNA template.
• Holoenzyme is made up of 𝛔-subunit plus the core enzyme. The 𝛔- subunit (‘sigma factor ) helps the RNA polymerase to recognize the promoter region on the DNA. 

Promoter region

In E.coli, RNA polymerase binds within a region stretching from about 70 base pairs (bp) before the transcription starts sites to about 30 bp after it. The first base at the transcription start site is “+1,” but none of the bases is designated “0”.

The promoter region extends between compositions -70 and +30. The 𝛔-subunit in the RNA polymerase recognizes the promoter site. The sequences that the sigma factor recognizes in the promoter site are called the Pribnow box.  

Pribnow box is located to the left of the transcription start site at -10 regions. The six nucleotides (5’- TATAAT– 3’ or TATA box) are present in it. 

-35 sequence is located at 35 bases to the left of the transcription start site. The nucleotide sequence (5’-TTGACA-3’) is present in it.

Binding

RNA polymerase recognizes and binds the promoter region in the DNA. Initially, it forms the closed complex in which the DNA is intact. Later it forms the open complex in which DNA starts to unwind with the action of the RNA polymerase enzyme. Unwinding occurs near the -10 sequence or the TATA box because there are no sufficient GC nucleotide pairs, and has weak bonding in this region. Separation of the two DNA strands takes place over 17 bp segments. The bases present in the coding strand are exposed, allowing the synthesis of the RNA by adding the ribonucleoside triphosphates.

Elongation

RNA polymerase starts to synthesize a transcript of the DNA sequence and does not need primer either. Usually, purine ribonucleotide is added in the beginning. As the RNA strand synthesizes, an RNA-DNA hybrid helix (8-9 nucleotides) is formed. The total structure is called a transcription bubble. The sigma subunit is released after the formation of the DNA-RNA hybrid. 

The polymerase leaves the promoter and functions to elongate the RNA. The sigma subunit needs to be released to elongate the RNA because it blocks the RNA exit channel. Then core enzyme of the RNA polymerase moves along the DNA template to the termination point for the elongation of the RNA. To join the ribonucleotides, a phosphodiester bond is formed. The ribonucleotides Adenine (A), Uracil (U), Guanine (G), and Cytosine (C) are added to the growing chain. Pyrophosphate is released whenever a ribonucleotide is added. DNA topoisomerases I and II relax the supercoils which may be formed during the elongation process. 

Termination

RNA chain keeps on elongating until it reaches the termination point. There are two types of termination: rho (⍴)  dependent termination and rho independent termination. 

Rho-dependent termination

Rho (⍴) protein is required to release the RNA product. Rho protein neither binds to the RNA polymerase nor the DNA. It binds tightly to RNA, where it reaches the RNA polymerase-DNA complex. Rho is an ATP-dependent RNA-stimulated helicase that disrupts the nascent RNA-DNA complex. Rho protein binds the C-rich region near the 3’ end of the newly synthesized RNA and migrates till it finds the termination site in the 5’-3’ direction.  

When it reaches the termination site, RNA and RNA polymerase are released from the DNA. Hence the transcription is terminated in it.

Rho-independent termination

It does not require rho protein. The RNA polymerase can recognize termination signals, i.e., GC-rich sequence on the DNA template. It consists of the inverted repeat with the central non-repeating segment.  

In the newly formed RNA, intra-strand base pairing occurs by folding back on themselves. Then it forms the hairpin loop-like structure. The A sequences follow this loop structure in the DNA template. So the complementary base pair in the RNA will be U sequences in the RNA. The binding of the UA is weak because it contains only two hydrogen bonds in U=A. Similarly, at the base of the stem, the GC-rich region stabilizes the secondary structure of the hairpin. 

When the synthesis of the RNA molecule is terminated, the enzyme separates from the DNA template. It then dissociates to the free core enzyme and free rho factor. 

 Difference between transcription in prokaryotes and eukaryotes

Prokaryotic transcription	Eukaryotic transcription
Occurs in the cytoplasm	Occurs in the nucleus
Only one RNA polymerase can synthesize all 3 types of RNA (mRNA, rRNA, tRNA )	Three RNA polymerases, I, II, and III, synthesize rRNA, mRNA, and tRNA, respectively.
Initiation of transcription does not need any initiation factors or proteins.	Initiation of transcription requires proteins called transcription factors ( TFIIA, TFIIB, TFIIID, TFIIE, TFIIF, and TFIIH) which recognize the TATA box.
RNA polymerases are complexes of 5-polypeptides.	RNA polymerases are complexes of 10-15 polypeptides.
Transcriptional unit has one or more genes (polycistronic)	Transcriptional unit has only one gene (monocistronic).
Inhibitors: Rifampin: inhibits binding of RNA polymerase Actinomycin: interrupts the movement of RNA polymerase	Inhibitors: α-amanitin: inhibits RNA polymerase II.

References: 

• Madigan, M. T., Martinko, J. M., Stahl, D. A., & Clark, D. P. (2011). BROCK Biology of Microorganisms (13th edition). Benjamin Cumming.