Transcription

BBM FK UNTAR
 Transcription
Transcription is the first step of gene
expression, in which a particular segment
of DNA is copied into RNA by the enzyme
RNA polymerase.

The synthesis of RNA molecules using

DNA strands as the templates so that
the genetic information can be
transferred from DNA to RNA.
 Differences between
replication and transcription

replication transcription

template double strands single strand

substrate dNTP NTP

primer yes no

Enzyme DNA polymerase RNA polymerase

product dsDNA ssRNA

base pair A-T, G-C A-U, T-A, G-C

 Similarity between
replication and transcription

• Both processes use DNA as the

template.
• Phosphodiester bonds are formed in
both cases.
• Both synthesis directions are from 5´
to 3´.
• The synthesis of an RNA molecule from DNA is a complex process involving one of
the group of RNA polymerase enzymes and a number of associated proteins.

• The general steps required to synthesize the primary transcript are:

initiation,
elongation, and
termination.

Most is known about initiation. A number of DNA regions (generally located upstream
from the initiation site) and protein factors that bind to these sequences to regulate the
initiation of transcription have been identified.

Certain RNAs—mRNAs, in particular—have very different life spans in a cell. It is

important to understand the basic principles of mRNA synthesis and metabolism, for
modulation of this process results in altered rates of protein synthesis and thus a
variety of both metabolic and phenotypic changes.

This is how all organisms adapt to changes of environment. It is also how

differentiated cell structures and functions are established and maintained.

The RNA molecules synthesized in mammalian cells are made as precursor

molecules that have to be processed into mature, active RNA. Errors or changes in
synthesis, processing, and splicing of mRNA transcripts are a cause of disease.
 Types of RNA Molecules
• Messenger RNAs (mRNAs)—intermediates that carry genetic
information from DNA to the ribosomes
• Transfer RNAs (tRNAs)—adaptors between amino acids and the
codons in mRNA.
• Ribosomal RNAs (rRNAs)—structural and catalytic components of
ribosomes.
• Small nuclear RNAs (snRNAs)—s tructural components of
spliceosomes.
• Micro RNAs (miRNAs)—short single-stranded RNAs (20 to 22 bp) that
block expression of complementary mRNAs.
• RNAi is similar to miRNA (RNA interference, double strand RNA,
plant) siRNA (small interference)
• piRNA (Piwi-interacting RNA) is a small non-coding RNA
molecules

© John Wiley & Sons, Inc.

 Eukaryotic RNA Polymerases and General Transcription
Factors
Although transcription proceeds by the same fundamental mechanisms in
all cells, it is considerably more complex in eukaryotic cells than in
bacteria. This is reflected in two distinct differences between the
prokaryotic and eukaryotic systems.

First, whereas all genes are transcribed by a single RNA polymerase in

bacteria, eukaryotic cells contain multiple different RNA polymerases that
transcribe distinct classes of genes.

Second, rather than binding directly to promoter sequences, eukaryotic

RNA polymerases need to interact with a variety of additional proteins to
specifically initiate transcription. This increased complexity of eukaryotic
transcription presumably facilitates the sophisticated regulation of gene
expression needed to direct the activities of the many different cell types of
multicellular organisms © John Wiley & Sons, Inc.
 Classes of Genes Transcribed by Eukaryotic RNA Polymerases.

a Some small nuclear (sn) and small cytoplasmic (sc) RNAs are transcribed by
polymerase II and others by polymerase III.
b The mitochondrial and chloroplast RNA polymerases are similar to bacterial
enzymes.
RNA Is Synthesized from a DNA Template
by an RNA Polymerase

• The processes of DNA and RNA synthesis are similar in that they involve
(1) the general steps of initiation, elongation, and termination with 5' to 3' polarity;
(2) large, multicomponent initiation complexes; and
(3) adherence to Watson-Crick base-pairing rules.

• These processes differ in several important ways, including the following:

(1) ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides;
(2) U replaces T as the complementary base pair for A in RNA;
(3) a primer is not involved in RNA synthesis;
(4) only a portion of the genome is transcribed or copied into RNA, whereas the
entire genome must be copied during DNA replication;
(5) there is no proofreading function during RNA transcription.
Watson and Crick model of the double-helical structure of the B form of DNA.
Messenger RNA
The relationship between the sequences of an RNA transcript and its gene, in which
the coding and template strands are shown with their polarities.

The RNA transcript with a 5' to 3' polarity is complementary to the template strand
with its 3‘ to 5' polarity. Note that the sequence in the RNA transcript and its polarity
is the same as that in the coding strand, except that the U of the transcript replaces
the T of the gene.
 The Template Strand of DNA Is Transcribed

• The strand that is transcribed or copied into an RNA molecule is referred to as the
template strand of the DNA. The other DNA strand, the non-template strand, is
frequently referred to as the coding strand of that gene. It is called this because,
with the exception of T for U changes, it corresponds exactly to the sequence of the
RNA primary transcript, which encodes the (protein) product of the gene.

• In the case of a double-stranded DNA molecule containing many genes, the template
strand for each gene will not necessarily be the same strand of the DNA double helix
Thus, a given strand of a double-stranded DNA molecule will serve as the template
strand for some genes and the coding strand of other genes.

Note that the template strand is always read in the 3' to 5' direction.
The Prokaryotic Transcription

• DNA-Dependent RNA Polymerase Initiates Transcription at a Distinct Site, the

Promoter

• E coli RNAP consists of a core complex of : α2ββ’ω, often termed E, associates

with a spesific protein factor (sigma σ factor) to form holoenzyme, α2ββ’ωσ or Eσ

• The transcription "bubble" is an approximately 20-bp area of melted DNA, and the
entire complex covers 30–75 bp, depending on the conformation of RNAP.
The transcription cycle in bacteria
Bacterial RNA transcription is described in five steps:
(1) Template binding: RNAP binds to DNA and locates a promoter (P) melts
the two DNA strands to form a preinitiation complex (PIC).

(2) Chain initiation: RNAP holoenzyme (core + one of multiple σ factors)

catalyzes the coupling of the first base (usually ATP or GTP) to a second
ribonucleoside triphosphate to form a dinucleotide.

(3) Promoter clearance: RNAP undergoes a conformational change after RNA

chain length reaches 10–20 nt and then is able to move away from the
promoter, transcribing down the transcription unit.

(4) Chain elongation: Successive residues are added to the 3'-OH terminus of
the nascent RNA molecule.

(5) Chain termination and release: The completed RNA chain and RNAP are
released from the template. The RNAP holoenzyme re-forms, finds a
promoter, and the cycle is repeated.
Bacterial promoters, E Coli

• TATA Box ( Pribnow Box)

• The Fidelity & Frequency of Transcription Is Controlled by Proteins Bound to

Certain DNA Sequences.

• Bacterial Promoters Are Relatively Simple

Rho-dependent transcription termination signals

• Bacterial transcription termination signal contains: inverted repeat ( dyad symetry)

(the two boxed areas) followed by a stretch of AT base pairs.

• The inverted repeat, when transcribed into RNA, can generate the hairpin secondary
structure in the RNA transcript. Formation of this RNA hairpin causes RNAP to pause
and subsequently the termination factor interacts with the paused polymerase and
somehow induces chain termination.

• Transcription continues into the AT region, and with the aid of the ρ termination
protein the RNA polymerase stops, dissociates from the DNA template, and releases
the nascent transcript.
Rho-dependent transcription termination signals

• Bacterial transcription termination signal contains: inverted repeat ( dyad symetry)

(the two boxed areas) followed by a stretch of AT base pairs.

• The inverted repeat, when transcribed into RNA, can generate the hairpin secondary
structure in the RNA transcript. Formation of this RNA hairpin causes RNAP to pause
and subsequently the termination factor interacts with the paused polymerase and
somehow induces chain termination.

• Transcription continues into the AT region, and with the aid of the ρ termination
protein the RNA polymerase stops, dissociates from the DNA template, and releases
the nascent transcript.
Eukaryotic Promoters Are More Complex
Schematic diagram showing the transcription control regions in a hypothetical
mRNA-producing, eukaryotic gene transcribed by RNA polymerase II. Such a gene
can be divided into its coding and regulatory regions, as defined by the transcription
start site (arrow; +1).

Proximal and distal cis elements are bound by trans -acting transcription factors, in
this example: Sp1 and CTF (also called C/EBP, NF1, NFY). These cis elements can
function independently of orientation (arrows).
 Basal Transcription Complex

• Formation of the basal transcription complex begins when TFIID binds to the TATA
box. It directs the assembly of several other components by protein-DNA and
protein-protein interactions; TFIIA, B, E,F, H, and polymerase II (pol II).
The entire complex spans DNA from position –30 to +30 relative to the initiation site
(+1, marked by bent arrow)
Promoter Accessibility
and Hence PIC
Formation
Is Often Modulated
by Nucleosomes

Nucleosome eviction by
chromatin-active coregulators
facilitates PIC formation and
transcription.
Transcription Factors
Two Models Can Explain the Assembly of the
Preinitiation Complex
• RNA MOLECULES ARE USUALLY PROCESSED BEFORE THEY BECOME
FUNCTIONAL

• The Coding Portions (Exons) of Most Eukaryotic Genes Are Interrupted

by Introns

• Introns Are Removed & Exons Are Spliced Together

• Alternative Splicing Provides for Different mRNA

• Alternative Promoter Utilization Provides a Form of Regulation

• Messenger RNA (mRNA) Is Modified at the 5' & 3' Ends

 A. Stepwise B. Holoenzyme

TFIID, TFIIA, TFIIB, TFIIE, TFIIH, TFIIF, Med Binding of a single protein complex:
pol II, Med and six GTFs
 RNA Pol I & III recognize distinct
The transcription in eukaryotes

promoters , using distinct sets of

transcription factors, but still require
TBP

• Pol I: transcribes rRNA precursor encoding gene

(multi-copy gene)
• Pol III: transcribes tRNA genes, snRNA genes and
5S rRNA genes

91
 Pol I promoter recognition

Upstream control element

UBF binds to the upstream of UCE, bring SL1 to the downstream part of UCE. SL1 in
turn recruits RNAP I to the core promoter for transcription

Fig 12-21 Pol I promoter region 92

 Pol III promoter recognition
1. Different forms, 2. locates downstream of the
transcription site

TFIIIC binds to the promoter, recruiting TFIIIB,

which in turn recruits RNAP III

93
Fig 12-22 Pol III core promoter
(3)RNAP I and III transcription
---GTFs and promoter recognition, formation of the
initiation complex

94
Key points of the chapter

1.RNA polymerases (RNAP) and transcription cycle

(Initiation is more complicate, details in bacteria)
2.Transcription cycle in bacteria:
(1) promoters (elements), s factor (4 domains),
aCTD, abortive initiation (why?)
(2) Structures accounting for formation of the
closed complex, transitions to open complex and
then stable ternary complex.
(3) Elongation and editing by polymerase (10-4)
(4) Termination: Rho-independent and Rho-
dependent mechanism
3.Transcription cycle in eukaryotes:
(1)Promoters (elements), general transcription
factors (GTF),
(2)RNAP II transcription
---the roles of GTFs and the CTD tail of RNAP II
in promoter recognition, formation of the pre-
initiation complex, promoter melting, promoter
escape
---in vivo requires mediator complex, nucleosome
modifying enzymes and transcription regulatory
proteins.
---elongation and proofreading involve a new set
of GTFs (What)
---coupled with RNA processing (How)