Transcription
Objectives
At the end of this
lecture, students will be able to –
• Discuss the process of transcription in
– Prokaryotes
– Eukaryotes
• Explain post transcriptional modification of mRNA
• Explain the mechanism, functions, and various classes of
transcription factors
Content
• Transcription in Prokaryotes
• Transcription in Eukaryotes
Transcription factors
• Sequence specific DNA binding factors
• Protein that binds to specific DNA sequence
• Control the flow of genetic information from DNA to mRNA
• They perform function alone or with other protein in
complex
• Act either by promoting or blocking the recruitment of RNA
polymerase to specific gene
Protein
Synthesis
• Process in which cells build proteins from information in
a DNA gene in a two major steps:
I-Transcription and
II-Translation
• Transcription:
Synthesis of an RNA (mRNA) that is complementary to one of the strands of DNA
• Translation:
Ribosomes read a messenger RNA and make protein according to its instruction
Transcription
• RNA polymerase copies both the exons and the introns.
• Stretch of DNA that is transcribed into an RNA molecule a
transcription unit
• A transcription unit contains coding sequence that is
translated into protein and sequences that direct and regulate protein
synthesis
• Transcription proceeds in the 5′ → 3′ direction
Structure
of RNA polymerase
• The holoenzyme is a complete RNA polymerase consisting of
an core enzyme and a sigma factor
• The core enzyme consists of 5 polypeptide chains
• Two α subunits, one β and β1 subunit and ώ subunit, σ factor
Functions
of RNA polymerase
• Having helicase activity for unwinding.
• Not requires primer
• Lacks proof reading
• σ – Recognition of promoter with the help of transcription
factors
• α – activator
• β – phosphodiester bond
• β – DNA template
RNA
polymerase
• RNA polymerase;
in prokaryotes only the single enzyme
• RNA polymerase governs the synthesis of all cellular RNAs
• In eukaryotic nuclei contain three RNA polymerases
• RNA polymerase I is found in the nucleolus
• The other two polymerases are located in the nucleoplasm
• The three nuclear RNA polymerase have different roles In
transcription
• Polymerase I makes a large precursor to the major rRNA
(5.8S, 18S and 28S rRNA in vertebrates)
• Polymerase II synthesizes hnRNAs, which are precursors to
mRNAs and small nuclear RNAs (snRNAs)
• Polymerase III synthesize the precursor to 5SrRNA, the
tRNAs and several other small cellular and viral RNAs
Initiation
in prokaryotes
Promoter recognition:
• Sigma factor interacts with core enzyme at β subunit site
to check transcription of both the strands by core enzyme
• The holoenzyme transcribes only one of two strands.
• Sigma factor of holoenzyme recognizes the promoter region
of the DNA
Promoters
in prokaryotes
Centered at -10 to -35 bp from the transcription start point
Promoters in
eukaryotes: 3 different promoters
-25 -40 -110
T A T A GC CAAT
• Binding of RNA
polymerase: Promoters have binding site for proteins rather than RNA
polymerase.
• Most common binding site – a complex of cyclic AMP
receptor protein
• Unwinding of DNA
double helix: Binding of ώ factor results in unwinding of a double helix
• Open complex allows tight binding of the RNA polymerase
with subsequent initiation of RNA synthesis
Synthesis
of first base of RNA chain
• The base of RNA synthesized is always in the form of
purine i.e. triphosphate guanine (ppp G) or adenine (ppp A)
• Initiation of mRNA synthesis does not require primer
Initiation ends after the formation of first inter nucleotide bond.
Elongation
• Core enzyme moves along the template from 3ꞌ-5ꞌ end untwisting the helix bit by bit and adding
one complementary nucleotide
• After 8-9 bp of RNA synthesis occurs, sigma factor is
released and recycled for other reaction
• RNA polymerase completes the transcription at 30-50 bp/sec
• Unwinding and rewinding of DNA occurs simultaneously
Termination
Two types of terminator sequence occur in prokaryotes
1. Type 1(ρ-
independent):
• RNA molecule terminated without the aid of the rho factor
contain GC rich sequence followed by U residues
• GC region makes RNA to spontaneously fold into hairpin
loop that tends to pull the RNA away from DNA
• The weaker bonds between the sequence of U residues and DNA template broken releasing
the newly formed RNA molecule
2. Type 2 (rho-
dependent):
• RNA molecule that do not form GC rich hairpin loop
requires rho factor for Termination
• It is a hexameric factor which binds to specific
termination sequence 50-90 bases located near 3ꞌ
end of newly forming RNA molecule
• It acts as an ATP – dependent unwinding enzyme, unwinds RNA
from DNA template as it proceeds
Transcription in eukaryotes Initiation
• RNA polymerase cannot recognize the promoters, requires
general transcription factor (GTFs)
• Promoters forms pre intiation complex with GTFs
• Assembly of the proteins to TATA box forms TATA binding
protein (TBP)
• TBP is present as a subunit of much larger protein complex
called TFІІD which specially binds to TATA box
Initiation
• TFІІB provide a binding site for RNA polymerase
• TFІІF contains subunit homologous to the bacterialσ
factor, bounds to the entering polymerase
• TFІІH contains 10 subunits, 3 possess enzymatic activity
helps in unwinding the DNA (helicase activity)
Elongation
• Same as prokaryotes
• Involves sequential addition of nucleotide units
Termination:
• Transcription by RNA polymerase І is terminated by a
protein factor that recognizes an 18-nucleotide termination signal
• Termination signals for RNA polymerase III include short
run of Us (as in prokaryotic signal). No proteins factors are needed for their
recognition
Post
transcription processing of mRNA
• Post-transcriptional modification is a process by which,
in eukaryotic cells, primary transcript RNA is converted into mature RNA
• Conversion of precursor messenger RNA into mature
messenger RNA (mRNA), which includes splicing and occurs prior to protein
synthesis
• The pre-mRNA molecule undergoes three main modifications
• 5′ capping
• 3′ polyadenylation
• RNA splicing – occur in the cell nucleus before the RNA is
translated
• The 5′ capping:
5ꞌ end chemically
modified by the addition of 7 methylguanosine
• Replacement of triphosphate group at the 5′ end of the RNA
chain with a special nucleotide GMP nucleotide
3’ adenylation
• Addition of poly A tail to 3’ end
• Added before it leaves the nucleus
• AAUAA sequence recognized by a specific endonuclease that
cleaves the RNA around 20 nucleotide down stream
• Poly A tail associated with protein, retard action of 3’-
exonucleases
Splicing
• RNA splicing – introns are removed from the pre-mRNA
• Remaining exons connected to re-form a single continuous
molecule
• Catalyzed by a large protein complex called the spliceosome
• Allows production of a large variety of proteins from a
limited amount of DNA
Mechanism
of transcription factors
• Stabilize or block the binding of RNA polymerase to DNA
• Catalyse acetylation or deacetylation of histone
• Histone acetyl transferase activity – acetylates histone
proteins
• Histone deacetylases activity – decetylates histone
proteins
• Recruit coactivators or corepressor proteins to the
transcription factor-DNA complex
Functions
of transcription factors
• Reads and interprets the genetic “blue print” in the DNA
• Bind to DNA and initiate program of increase or decrease gene
transcription
• Basal transcription regulation
– General transcription factors necessary for transcription
to occur
– They interact with polymerase directly
Functions
of transcription factors
Differential
enhancement of transcription
• Regulate the expression of various gene by binding to
enhancer region of DNA adjacent to regulated gene
• Ensure that genes are expressed in right cell at the right
time
Response to
intracellular cells
• Transcription factors are involved in the downstream of
signalling cascade
Response to
environment
• Involved in downstream of signaling cascade in
environmental stimuli
• Heat shock factors, hypoxia inducible factors
Cell cycle control
• Proto-oncogenes or tumor suppressor gene regulate cell
cycle
• Myc oncogene – in cell growth & apoptosis
Regulation
of transcription factors
Synthesis
• Transcription factors are transcribed from gene on a
chromosome into RNA, then RNA to proteins
• Any of these steps can be regulated to affect the
production of transcription factors
Nuclear localization
• Transcribe in nucleus but not translated in cytoplasm
• They have nuclear localization signals that direct them to
nucleus
Activation
• Transcription factors can be activated or inactivated by
through signal sensing domain
• Ligand binding –
influence factors present in cell; decided if factors are in active state or
capable to bind DNA
• Phosphorylation –
STAT protein must be phosphorylated before they can bind to DNA
• Interaction with other transcription factors
Accessibility of DNA
binding site
• DNA in nucleosome is inaccessible to many transcription
factors
• Nucleosome should be actively removed by molecular motors
Availability of other
cofactors/ transcription factors
• Most transcription factors do not work alone
• For transcription many factors must bind to DNA regulatory
sequence
• Recruitment of intermediary proteins
Classes of
transcription factors
Classified based on –
• Mechanism of action
• Regulatory function
• Sequence homology in their DNA binding domain
Mechanistic
class of transcription factor
• General
transcription factors
– Involved in the formation of pre initiation complex
– TFIIA, TFIIB, TFIID, TFIIE, TFIIF
– Ubiquitous, interact with core promoter region
• Upstream
transcription factors
– Binds upstream to initiation site
– Stimulate or repress transcription
Functional
class of transcription factors
Constitutively active
• Present in all cells at all time – general transcription
factors Sp1, NF1, and CCAAT
Conditionally active
– requires activation
• Developmental – cell specific
• Signal – dependent – requires external signal for
activation
Structural
class of transcription factor
• Based on sequence similarity and tertiary structure of
DNA- binding domain
• 1- superclass – Basic domains
• 2 – Superclass – Zinc co-ordinating DNA binding domain
• 3 Superclass – Helix-turn-helix
• 4 superclass- beta-scaffold factors with minor groove
contacts
• 5 superclass – other transcription factors
Summary
• Protein synthesis in prokaryotes and eukaryotes occurs
through two steps – Transcription and Translation
• Process of transcription involves 3 steps – initiation,
elongation and termination
• Process of transciption in similar in prokaryotes and
eukaryotes but differs in post transcriptional modification
• Post transcriptional modifications include addition of 5’
cap, 3’ adenylation and RNA splicing
• Transcription factors are sequence specific DNA binding factors
and protein that binds to specific DNA sequence
• Stabilize or block the binding of RNA polymerase to DNA
• They reads and interprets the genetic “blue print” in the
DNA
• Also bind to DNA and initiate program of increase or
decrease gene transcription
• Transcription factor is classified based on mechanism of
action, regulatory function and sequence homology