Transcription- a short approach for UG zoology students | Study notes Molecular biology

CORE COURSE 2: Molecular Biology

Unit 3: Transcription

Transcription is the process through which a DNA sequence is enzymatically copied by an RNA polymerase to

produce a complementary RNA. The synthesis of RNA from a single strand of a DNA molecule in the presence of

enzyme RNA polymerase is called transcription. It is the process of formation of a messenger RNA molecule using

a DNA molecule.

Transcription in Prokaryotes:

➢ Transcription in prokaryotes (and in eukaryotes) requires the DNA double helix to partially unwind in the

region of mRNA synthesis.

➢ RNA synthesis in the nucleus was exported to the cytoplasm.

➢ The region of unwinding is called a transcription bubble.

➢ Transcription always proceeds from the same DNA strand for each gene, which is called the template

strand.

➢ The mRNA product is complementary to the template strand and is almost identical to the other DNA

strand, called the non template strand.

➢ The only difference is that in mRNA, all of the T nucleotides are replaced with U nucleotides. In an RNA

double helix, A can bind U via two hydrogen bonds, just as in A–T pairing in a DNA double helix.

➢ The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5' mRNA

nucleotide is transcribed is called the +1 site, or the initiation site.

➢ Nucleotides preceding the initiation site are given negative numbers and are designated upstream.

Conversely, nucleotides following the initiation site are denoted with “+” numbering and are called

downstream nucleotides.

➢ Upstream refers to “towards the 5’ end” and downstream refers to “towards the 3’ end”.

➢ The region of DNA that contains sequences that are the signals for transcribing a gene are termed

promoters.

➢ +1 refers to the basepair where transcription starts; -x refers to x basepairs 5’ to the start site.

Factors required for transcription:

1. RNA polymerase (enzyme that catalyzes the synthesis of RNA from a DNA template):

➢ Prokaryotes use the same RNA polymerase is composed of five polypeptide subunits, two of

which are identical.

➢ Four of these subunits, denoted α, α, β, and β' comprise the polymerase core enzyme.

➢ These subunits assemble every time a gene is transcribed, and they disassemble once transcription

is complete.

➢ Each subunit has a unique role; the two α-subunits are necessary to assemble the polymerase on

the DNA; the β-subunit binds to the ribonucleoside triphosphate that will become part of the

nascent “recently born” mRNA molecule; and the β' binds the DNA template strand.

➢ The fifth subunit, σ, is involved only in transcription initiation. It confers transcriptional

specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation

site.

➢ Without σ, the core enzyme would transcribe from random sites and would produce mRNA

molecules that specified protein gibberish. The polymerase comprised of all five subunits is called

the holoenzyme.

➢ Holoenzyme = core +  factor (recognizes the promoter)

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➢ RNA synthesis in the nucleus was exported to the cytoplasm.

1. RNA polymerase (enzyme that catalyzes the synthesis of RNA from a DNA template):

➢ Prokaryotes use the same RNA polymerase is composed of five polypeptide subunits, two of

which are identical.

➢ Four of these subunits, denoted α, α, β, and β' comprise the polymerase core enzyme.

➢ These subunits assemble every time a gene is transcribed, and they disassemble once transcription

is complete.

➢ Each subunit has a unique role; the two α-subunits are necessary to assemble the polymerase on

the DNA; the β-subunit binds to the ribonucleoside triphosphate that will become part of the

nascent “recently born” mRNA molecule; and the β' binds the DNA template strand.

➢ The fifth subunit, σ, is involved only in transcription initiation. It confers transcriptional

specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation

site.

➢ Without σ, the core enzyme would transcribe from random sites and would produce mRNA

molecules that specified protein gibberish. The polymerase comprised of all five subunits is called

the holoenzyme.

➢ Holoenzyme = core +  factor (recognizes the promoter)

2. Accessory transcription activator proteins:

➢ Can bind to specific DNA sequences and help RNA polymerase initiate transcription via protein-

protein interactions or by altering the structure of the DNA.

➢ Transcription of some promoters requires an accessory transcriptional activator; at other

promoters, the activators just increase the rate of transcription but are not absolutely required.

3. Template DNA containing gene or genes to be transcribed:

➢ The RNA is synthesized from a single template [strand] of a DNA molecule. The stretch of DNA

that is transcribed into an RNA molecule is called a transcription unit.

➢ A transcription unit codes the sequence that is translated into protein. It also directs and regulates

protein synthesis.

➢ The DNA strand which is used in RNA synthesis is called template strand; because it provides the

template for ordering the sequence of nucleotides in an RNA transcript.

➢ The DNA strand which does not take part in DNA synthesis is called coding strand, because, its

nucleotide sequence is the same as that of the newly created RNA transcript.

➢ The regulatory element that determine when a gene “turned on” (transcribed) or “turned off”.

➢ The promoter DNA is located upstream of the gene and contains a sequence which  factor of

RNAP and other transcription factors bind.

➢ The specific sequence of a promoter is very important because it determines whether the

corresponding gene is transcribed all the time, some of the time, or infrequently.

➢ Although promoters vary among prokaryotic genomes, a few elements are conserved.

➢ At the - 10 and - 35 regions upstream of the initiation site, there are two promoter consensus

sequences, or regions that are similar across all promoters and across various bacterial species.

➢ The - 10 consensus sequence, called the - 10 region, is TATAAT. The - 35 sequence, TTGACA , is

recognized and bound by σ.

➢ Once this interaction is made, the subunits of the core enzyme bind to the site.

➢ The A–T-rich - 10 region facilitates unwinding of the DNA template, and several phosphodiester

bonds are made.

 factor of RNAP binds the corresponding  factor recognition sequence in the promoter.

RNAP is bound covering approx. 60 basepairs. The DNA is still is a double helix (closed complex).

RNAP unwinds the DNA resulting in open complex formation.

First nucleotides are added to start RNA chain. Transcriptional initiation has occurred

2. Elongation:

In the prokaryotes, the elongation starts with the “abortive initiation cycle”. During this cycle RNA

Polymerase will synthesize mRNA fragments 2-12 nucleotides long. This continues to occur until the σ

factor rearranges, which results in the transcription elongation complex (which gives a 35 bp moving

footprint). The a factor is released before 80 nucleotides of mRNA are synthesized.

❖ After synthesis of RNA more than 10 bp long, the σ-factor is dissociates and the enzyme move

along 5’-3’ direction continuously synthesizing RNA.

❖ The synthesized RNA exit from RNA exit channel.

❖ The synthesized RNA is proof reads by Hydrolytic editing. For this the polymerase back track by

one or more nucleotide and cleave the RNA removing the error and synthesize the correct one.

The Gre factor enhance this proof reading process.

❖ Pyrophospholytic editing another mechanism of removing altered nucleotide.

Fig: During elongation, the prokaryotic RNA polymerase tracks along the DNA template, synthesizes mRNA in the

RNA Polymerase I: