Transcription

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 3ꞌend 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