Transcription

In this lesson, you will be introduced to the structure of a gene, RNA polymerase core enzyme, and the
different events in transcription. After this, the molecular details of the transcription initiation and
termination will be elaborated.

Jayzon G. Bitacura, MSc., RMicro

Assistant Professor III
Department of Biological Sciences
College of Arts and Sciences
Visayas State University
 Learning Outcomes
At the end of this lesson, you will be able to:
1. describe the structure of a gene.
2. describe the structure and function of RNA Polymerase core enzyme.
3. discuss the events in transcription.
4. identify the different initiation factors in Prokaryotes and Eukaryotes; and
5. differentiate the termination process in Prokaryotes and Eukaryotes.

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Motivation
Transcribe the following DNA segment.

3’ T A C G G C T T A C C A G A T G C G C G T A T T 5’
5’ A T G C C G A A T G G T C T A C G C G C A T A A 3’

___________________________________________________

You are correct! The mRNA produced after transcription of the given DNA
segment is

5’ A U G C C G A A U G G U C U A C G C G C A U A A 3’

How does the cell able to synthesize an RNA

molecule?

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Transcription
● Transcription is the process wherein
genetic information from DNA is
transferred into RNA.
● It is the first step in the expression of
the genetic information stored in the
genome: from gene to gene product
(frequently a protein).
● It became very rapidly clear in the
50s that DNA could not be directly
converted into protein, and there had
to be an intermediate step.
● This unstable product was identified
as RNA.
● It is a single-stranded
molecule that contains Uracil
instead of Thymine.
● It uses ribose sugar instead
of deoxyribose.

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Classes of RNA
Three major classes of RNA:
1. Ribosomal RNA, rRNA
2. Transfer RNA, tRNA
3. Messenger RNA, mRNA
They participate in protein synthesis and are synthesized from DNA by
transcription

Other forms of RNA:

• small nuclear RNA (snRNA).
• small nucleolar RNA (snoRNA).
• microRNA (miRNA). ~22 nt RNA molecules that regulate the
expression of mRNA molecules.
• long non-coding RNA (lncRNA).
• siRNA – RNA interference >>> 2006 Nobel Prize Andrew Z. Fire &
Craig C. Mello

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The Structure of a Gene
● What is a gene? How does a gene look like? What are its
components?
● A gene is a segment (or segments) of the DNA with a distinct
sequence of nucleotides that is transcribed and translated into
a protein product that contributes to the expression of a specific
phenotype/function.
○ For example, a specific sequence in the DNA of humans
codes for the protein hemoglobin, which gives the
red-color phenotype to red blood cells.
○ Another sequence codes for the protein melanin,
responsible for the dark color phenotype of the skin and
hair.
● However, the protein product of a gene may also be an enzyme
responsible for a specific function, such as the enzyme amylase
that breaks down the starch in seeds resulting in seed
germination.

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The Structure of a Gene
● Genes of all organisms are composed of a promoter region, the RNA-coding sequence
or the open reading frame (ORF), and the terminator region.
● The sequences after the transcription initiation site towards the 3’ end of the template
strand are called upstream elements, while those sequences before the transcription
termination site towards the 5’ end are called the downstream elements.

http://web2.mendelu.cz/af_291_projekty2/vseo/print.p
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 The RNA Polymerase Core Enzyme
The CORE enzyme has five subunits (~400 kDa)
● β': largest subunit, encoded by rpoC.
Contains part of the active center responsible for RNA
synthesis
● β: second-largest subunit, encoded by rpoB.
Contains the rest of the active center responsible for
RNA synthesis.
● α: third-largest subunit and is present in two copies per
molecule of RNA pol, αI and αII. Each α contains two
domains: αNTD (N-Terminal domain) and αCTD
(C-terminal domain). αNTD required for assembly of
RNA pol.
αCTD required for interaction with the promoter,
non-sequence/non-specific interactions at most
promoters and sequence-specific interactions at
upstream-element-containing promoters. α interacts
with regulatory factors.
● ω: smallest subunit. ω facilitates assembly of RNAP
and stabilizes RNAP
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RNA polymerases in all living organisms are evolutionary
related

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Events in Transcription
To produce mRNA from a gene, RNA polymerase proceeds through a series of
well-defined steps grouped into three phases:
● Initiation
● Elongation
● Termination

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Events in Transcription: INITIATION
● During initiation, the RNA polymerase, together with any initiation factors required, binds to the
promoter, the DNA sequence where transcription starts.
● Once formed, the promoter-polymerase complex undergoes structural changes required for
initiation to proceed.
● The DNA will then unwind around the point where transcription will start.
● The base pairs are disrupted, producing a “transcription bubble” of single-stranded DNA.
● The new ribonucleotide is added to the 3’ end of the growing strand since transcription always
occurs in a 5’-to-3’ direction.

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● Only one DNA strand acts as a template on which the RNA strand is built.
● This strand is called the template strand or antisense strand or non-coding strand.
● The other strand which to where the code being copied is located is called the non-template
strand or sense strand or coding strand.
● Because RNA polymerase binds promoters in a defined orientation, the same strand is always
transcribed from a given promoter.
● The choice of promoter determines which stretch of DNA is transcribed and is the most common
step at which regulations are imposed.

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Events in Transcription: ELONGATION
● Once the RNA polymerase has synthesized a short stretch of RNA (~10 bases), it shifts into the
elongation phase.
● During elongation, the enzyme performs an impressive range of tasks in addition to the catalysis
of mRNA synthesis.
● It unwinds the DNA in front and reanneals it behind, it dissociates the growing mRNA chain from
the template as it moves along, and it performs proofreading functions.
● Recall that during replication, in contrast, several different enzymes are required to catalyze a
similar range of functions.

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Events in Transcription: TERMINATION
● Once the polymerase has finished transcribing the protein-coding region or the gene, it must stop
and release the mRNA product as well as dissociate from the DNA.
● In some cells, specific and well-characterized sequences signal termination of transcription.
● In others, it is less clear what triggers the enzyme to stop transcribing and detaching itself from
the template.

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The Promoter Region
● The prokaryotic promoter region has a
transcription start site (or initiation site) at -10
DNA region and a -35 DNA region.
● The -10 region is located 10 nucleotide (nt)
upstream of the transcription start site and is
known as the Pribnow-Schaller box.
● The -35 region is located at 35 nt upstream of
the transcription start site. Many prokaryotes
share a common or similar sequence at their -35
and -10 regions.
● These shared sequences are called consensus
sequences.
● At -10 region is the sequence TATAAT while at
-35 is the sequence TTGACA.
● The strength of a promoter is reflected by the
match between its -10 and -35 sequences and
the consensus sequences for these regions.
● Also, contact between the alpha subunits of
polymerase and the sequence between positions
-40 and -60 (UP elements) can contribute to
promoter recognition and strength.

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The Promoter Region
● The eukaryotic promoter region at -25 and -80 DNA regions upstream of the transcription start site.
● These regions also have consensus sequences. At -25 is the sequence TATA (TATA box) while at -80
is the sequence CAAT (CAAT box)

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Initiation Factors
● In prokaryotes, the initiation factor is called
the sigma factor (s).
● Sigma binds to the promoter region
recognizing both the -35 and -10 regions
forming a closed preinitiation complex.
● Sigma provides the helicase activity that
unwinds the DNA to form the open complex.
● Following initiation, sigma is released from
the holoenzyme after the first 10 nucleotides
have been polymerized.
● RNA polymerase containing sigma 70 is the
major polymerase species present during
exponential growth.

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Initiation Factors
● In eukaryotes, the initiation factor is called
a transcription factor (TF).
● The core promoter is where the assembly
of the transcription pre-initiation complex
(PIC) that includes TFIIA, TFIIB, TFIID,
TFIIE, TFIIF, TFIIH, and RNA pol II occurs.
● TATA-binding protein is also part of TFIID
that binds the TATA-box.

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Termination of Transcription in
Prokaryotes ● There are two main mechanisms for transcription termination in
prokaryotes:
● Rho-independent and Rho-dependent termination. How do they
differ?
● Rho-independent termination occurs when the termination
sequence forms a GC stem-loop in the nascent RNA, which causes
RNA pol to pause.
○ The stem-loop is followed by a stretch of U residues, which
destabilize the polymerization process and cause the
release of the RNA chain.
● Rho-dependent termination, the "hot pursuit" model for rho action
suggests that Rho binds to a nascent RNA chain.
○ This might require a specific sequence or not.
○ Rho then translocates along the mRNA.
○ The stretch of RNA-DNA hybrid in the transcription bubble
can be directly accessed by rho and cause it to unwind by its
helicase activity.
○ Completion of prokaryotic termination is distinguished by
the release RNA pol and rho from the nucleic acids.

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Termination of Transcription and RNA
processing in Eukaryotes
● Once transcribed, eukaryotic RNA has to be
processed in various ways before being
exported from the nucleus, where it can be
translated.
● These processing events include the capping of
the 5’ end of the RNA, polyadenylation of the 3’
end of the RNA, and splicing.

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Capping
● This process involves the addition of a modified
guanine base to the 5’ end of the RNA.
● Specifically, it is methylated guanine.
● A 7-methylguanosine cap is added to the 5' end of
the growing transcript by a 5'-to-5' phosphate linkage.
● The 5‘-cap protects the nascent mRNA from
degradation, promotes splicing, regulates nuclear
export, and assist ribosome binding during
translation.

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 ● Polyadenylation of the 3’ end of the mRNA is
intimately linked with the termination of
transcription.
Polyadenylation ● Once polymerase encounters the specific
sequences at the end of a gene after being
transcribed into RNA, the transfer of the
polyadenylation enzymes to the RNA is triggered.
● This leads to the occurrence of four events:
cleavage of the message, addition of many
adenine residues to its 3’ end, degradation of the
RNA remaining associated with RNA polymerase
by a 5’-to-3’ ribonuclease, and subsequently,
termination of transcription.
● The poly-A tail protects the mRNA that has just
been transcribed from degradation.
● It aids in the export of the mature mRNA to the
cytoplasm.
● Finally, it is involved in binding proteins
responsible for initiating translation.

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Splicing
● Unlike prokaryotes, eukaryotic genes have noncoding DNA sequences called introns that are
inserted between the coding sequences called exons.

● Thus, the final stage of RNA processing after the termination of transcription is the removal of
introns (noncoding sequences) and joining of the exons (coding sequences) together.

● This process is called splicing which is carried out in a series of reactions catalyzed by the
spliceosome, a ribonucleoprotein complex.

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Splicing
Splicing is a two-stage process:
● First is the formation of
2’,5’-phosphodiester bond between the
lariat site A in the intron and 5’
phosphate.
● And second, the free 3’-OH of 5’ exon
generates phosphodiester with 3’ exon,
which releases the intron lariat.
● The intron lariat is then debranched and
degraded.
● Take note that the transesterification
reactions do not require free energy
input.

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 Summary Questions

● What is transcription?
● What are the key players in the
transcription process?
● How does transcription occur? Can
you explain the molecular details of
each stages?

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Contact information:
jayzon.bitacura@vsu.edu.ph

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