DNA Transcription: Steps and Mechanism

Your genes are written in DNA, but DNA never leaves the safety of its storage to do the day-to-day work of the cell. Instead, the cell makes a working copy of a gene in the form of RNA, and it is this RNA copy that goes on to direct the building of proteins. That copying step, from DNA to RNA, is called transcription.

Transcription is the first step in turning a gene into a product, and it is the middle link of the central dogma of biology: DNA is copied into RNA (transcription), and RNA is read to build protein (translation). This article explains how transcription works in a clear sequence: which strand is copied, how the enzyme RNA polymerase starts, builds, and stops the RNA, and how the process differs between bacteria and higher organisms.

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 amino acids
• rRNA: component of the ribosomes

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

What is transcription?

Transcription is the process of making an RNA copy of a gene from a DNA template. The cell uses an enzyme called RNA polymerase to read one strand of the DNA and build a matching strand of RNA.

The rules of the copy are the base-pairing rules, with one change from DNA. Where the DNA template has A, the RNA gets U (uracil, which replaces thymine in RNA). Where the template has T, the RNA gets A. Where the template has G, the RNA gets C, and where it has C, the RNA gets G. So the RNA is complementary to the template strand.

Three important points before the steps:

1. RNA polymerase does not need a primer. Unlike DNA polymerase, it can start a new RNA strand from scratch. This is one key difference between transcription and replication.
2. Only one DNA strand is copied. For a given gene, RNA polymerase reads just one of the two DNA strands, called the template strand.
3. RNA is built in the 5′ to 3′ direction, reading the template 3′ to 5′, the same directional rule seen in replication.

Template strand versus coding strand

The two DNA strands have different roles in transcription. The template strand (also called the antisense or non-coding strand) is the strand that RNA polymerase actually reads to build the RNA. The coding strand (also called the sense or non-template strand) is the other strand; it is not read.

Figure: Template and coding strand Source:https://www.lecturio.com/concepts/stages-of-transcription/

Here is the useful part: the RNA that is made has the same sequence as the coding strand, except that U replaces T. That is why the coding strand is called "coding," even though it is not the one physically read. RNA polymerase reads the template strand, but the RNA it produces reads like the coding strand.

A worked example:

• Coding strand: 5′- A T G C -3′
• Template strand: 3′- T A C G -5′
• RNA made: 5′- A U G C -3′

Notice the RNA matches the coding strand (A T G C), with U in place of T. And it is complementary to the template strand, which is the strand RNA polymerase actually read.

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.

Figure: RNA polymerase: Holoenzyme

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 start site 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 region bound by RNA polymerase extends roughly from position -70 to +30, relative to the transcription start site. 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.

Figure: Pribnow box

Pribnow box is located to the left of the transcription start site at -10 regions. It contains a six-nucleotide sequence, 5′-TATAAT-3′.

-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

Figure: Binding of RNA polymerase, Source: Pearson Education

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 (the Pribnow box) because this AT-rich region has few GC pairs and therefore weaker bonding. Separation of the two DNA strands takes place over 17 bp segments.

The two strands separate, exposing the bases of the template strand. RNA polymerase reads this template strand and builds the RNA by adding ribonucleoside triphosphates one at a time.

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. As with replication, topoisomerase enzymes relax the supercoiling that builds up ahead of the moving RNA polymerase.

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.

Figure: Rho-independent termination

The newly formed RNA folds back on itself and forms a hairpin loop. Just after this hairpin, the template DNA has a run of adenines (A), so the RNA has a matching run of uracils (U). The U-A pairing here is weak, because each U-A pair has only two hydrogen bonds. The hairpin makes RNA polymerase pause, and the weak U-A pairing then lets the RNA break away from the template. 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.

Figure: Rho-independent termination Source: https://www.biolearners.com

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, TFIID, 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.

How to Remember

The central dogma place. Transcription is the first copy step: DNA to RNA. Translation is the second: RNA to protein. "Transcription writes the message, translation reads it." Both start with "t," so anchor by order: transcription comes first, alphabetically and biologically.

Template versus coding, settled once. RNA polymerase reads the template strand, but the RNA comes out matching the coding strand (with U for T). Memory line: "Read the template, copy the coding." The strand you read is not the strand the RNA looks like.

U replaces T. RNA has no thymine. Wherever DNA would put T, RNA puts U. "RNA is a U-turn from T."

The Pribnow box location. In bacteria, the key promoter sequence is the Pribnow box (TATAAT) at about the -10 position, ten bases before the start. "TATAAT at minus ten" ties the sequence to its place.

Two ways to stop. Bacterial transcription ends either with the rho protein (rho-dependent) or with a GC-rich hairpin that pops the RNA off (rho-independent). "Rho helps, or the hairpin does it alone."

Key exam facts in one table

Fact	Detail
What transcription does	Copies a gene from DNA into RNA
Enzyme	RNA polymerase
Needs a primer?	No (unlike DNA replication)
Strand read	Template strand (antisense)
RNA sequence matches	Coding strand (sense), with U in place of T
Base change from DNA	Uracil (U) replaces thymine (T)
Direction	RNA built 5′ to 3′
Three steps	Initiation, elongation, termination
Bacterial promoter	Pribnow box (TATAAT) at -10; also -35 sequence (TTGACA)
Termination (bacteria)	Rho-dependent, or rho-independent (GC-rich hairpin)
Prokaryote RNA polymerase	One enzyme for all RNA types
Eukaryote RNA polymerases	Three (Pol I, II, III) for different RNA types

Where Students Get Confused

Template strand versus coding strand. RNA polymerase reads the template (antisense) strand, but the RNA it makes matches the coding (sense) strand, with U instead of T. The strand that is read is not the strand the RNA resembles. This single point causes most transcription confusion; the worked example above is worth re-reading.

Transcription does not need a primer. DNA replication needs a primer to start, but transcription does not. RNA polymerase can begin a new RNA strand on its own. Do not carry the primer rule over from replication.

Transcription is not replication. Replication copies all of the DNA into DNA before cell division. Transcription copies one gene into RNA whenever that gene's product is needed. Different enzyme, different product, different purpose.

U replaces T, not the other way round. RNA uses uracil where DNA uses thymine. A common slip is to write thymine into RNA; RNA has no thymine.

The Pribnow box and the TATA box are related but not identical. The bacterial Pribnow box (TATAAT at -10) is the prokaryotic promoter element. Eukaryotes have a similar element called the TATA box. They play a similar role but are not the same sequence in the same organism.

References:

• Madigan MT, Bender KS, Buckley DH, Sattley WM, Stahl DA. Brock Biology of Microorganisms. 16th ed. Pearson; 2021.
• Watson JD, Baker TA, Bell SP, Gann A, Levine M, Losick R. Molecular Biology of the Gene. 7th ed. Pearson; 2013.
• Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 8th ed. W.H. Freeman; 2021.