21/10/62

Transcription & Transcription Post-transcriptional modifications • The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can 2019 be transferred from DNA to RNA

Chalermchai Mitrpant

Similarity between Differences between Replication and Transcription replication and transcription

replication transcription

• Both processes use DNA as the template template double strands single strand

• Phosphodiester bonds are formed in both substrate dNTP NTP cases primer yes no

• Both synthesis directions are from 5´ to 3´ Enzyme DNA polymerase RNA polymerase

product dsDNA ssRNA

base pair A-T, G-C A-U, T-A, G-C 21/10/62

Template DNA

• The whole genome of DNA needs to be replicated, but only small portion of Section 1 genome is transcribed in response to the development requirement, physiological need and environmental changes Template and Enzymes • DNA regions that can be transcribed into RNA are called structural genes

TRANSFAC Gene structure Strands of DNA

Regulatory Transcribed region Gene (Sense strand) Elements 5'G C A G T A C A T G T C 3' coding strand 3‘ 5‘ 3' C G T C A T G T A C A G 5' template Transcription primary strand Transcript (mRNA) (Antisense strand) transcription Splice variants Mature transcript Template for ribosome (mRNA) 5'G C A G U A C A U G U C 3' RNA 5’-UTR 3’-UTR (Sense strand) Translated region 21/10/62

Asymmetric transcription Organization of coding information in the genome

5' 3' 3' 5'

• Only the template strand is used for the transcription • The transcription direction on different strands is opposite

RNA polymerase RNA-pol of E. Coli

• The enzyme responsible for the RNA The holoenzyme of RNA-pol in E.coli consists synthesis is DNA-dependent RNA of 5 different core subunits:  plus  polymerase – The prokaryotic RNA polymerase is a multiple-subunit protein of ~480kD subunit MW function Determine the DNA to be  36512 – Eukaryotes have three kinds of RNA transcribed  150618 Catalyze polymerization polymerases, each of which is a multiple- Bind & open DNA  155613 subunit protein and responsible for template Recognize the promoter  70263 transcription of different RNAs for synthesis initiation 21/10/62

Eukaryotes have three nuclear RNAPs RNA-pol of E. Coli transcribe different classes of genes • Recognise promoter, a region of DNA involved in binding of RNA pol to facilitate assembly of the enzyme at transcription start site

• Utilise different sigma factor subunit to control different sets of gene to be transcribed • Each has 12‐17 different subunits

• 9 conserved subunits, 5 of which are related to subunits of bacterial RNAP

• Inhibitor ( subunit) of Bacteria RNA Pol II Mycobacterium Tuberculosis has been used for antimicrobial agent (Rifampicin)

Comparison of structure of RNA-pol How the Polymerase CTD Couples Transcription to between eukaryotes and prokaryotes other processes

Factors recruited Kinase/ phosphatase YSPTSPS capping factors TF II H, mediator P

elongation

pTEFb YSPTSPS splicing components (Cdk9) P In S. cerevisiae, shared by Cdk1 and Bur 1 P

phosphatases (Rtr1(2?) Further elongation YSPTSPS 3’ end processing factors P Termination Phosphatases (Fcp1, ssu72) YSPTSPS Mediator, activators 21/10/62

The RNA Pol II CTD is required for the coupling of transcription with posttranscriptional modifications Recognition of Origins

• Each transcriptable region is called operon • One operon includes several structural genes and upstream regulatory sequences (or regulatory regions) • The promoter is the DNA sequence that RNA-pol can bind. It is the key point for • The coupling allows the processing factors to present at high local concentrations when splice sites and poly(A) signals are transcribed the transcription control by Pol II, enhancing the rate and specificity of RNA processing

• The association of splicing factors with phosphorylated CTD also stimulates Pol II elongation. Thus, a pre-mRNA is not synthesized unless the machinery for processing it is properly positioned

Prokaryotic promoter Features of Some Promoters Recognized by 5' 3' Eukaryotic RNA Polymerase II -50 -40 -30 -20 -10 110 3' 5' -35 region -10 start region T T G A C A A A C T G T T A T A A T A T A T T A (Pribnow box) • The -35 region: Recognition site and the binding site of RNA-pol • The -10 region: Region in which stable complex of DNA and RNA-pol is formed 21/10/62

Regulatory sequences expand in number and complexity with increased complexity of the organism The TAFs in TFIID also serve as coactivators

~ 30‐100 bp

~ 100s bp

Could be 50kB or more

Regulatory control of eukaryotic transcription: interplay between enhancer and core promoter

Section 2

Transcription Process 21/10/62

General concepts §2.1 Transcription of Prokaryotes

• Three phases: initiation, elongation, and termination • Initiation phase: RNA-pol recognizes the promoter and starts the • The prokaryotic RNA-pol can bind to the transcription DNA template directly in the transcription • Elongation phase: the RNA strand is process continuously growing • The eukaryotic RNA-pol requires co- • Termination phase: the RNA-pol stops factors to bind to the DNA template synthesis and the nascent RNA is together in the transcription process separated from the DNA template

a. Initiation

Core RNAP Energy Steps in transcription initiation accumulation to remove sigma • RNA-pol recognizes the TTGACA region, factor and promoter escape and slides to the TATAAT region, then  slowing opens the DNA duplex. RNAP down • Step for initiation DNA unwinding – Promoter binding at the AT rich Short RNA chain sequence (‐ without moving – DNA Unwinding 9+3) from promoter NTPs K – RNA chain initiation B Kf Abortive Elongating R+P RPc RPo Initiation Complex initial “isomerization” binding 21/10/62

Validation of the prediction that occlusion of the Initial transcription involves DNA scrunching RNA exit channel promotes “abortive initiation”

• The energy accumulated in the DNA scrunched “stressed #1: transcription by holoenzyme with full-length #2: transcription by holoenzyme with truncated at Region 3.2: lacks in intermediate could disrupt interactions between RNAP, the RNA exit channel and the promoter, thereby driving the transition from

Murakami, Darst 2002 initiation to elongation (promoter escape) A. N. Kapanidis et al., Science 314, 1144 ‐1147 (2006)

Promoter escape b. Elongation

is positioned to affect key activities of RNA polymerase • The release of the  subunit causes the conformational change of the core enzyme the nascent RNA form a complex called the transcription bubble

• The 3 segment of the nascent RNA hybridizes with the DNA template, and its 5 end extends out the transcription bubble as the synthesis is processing 21/10/62

c. Termination The termination function of  factor

• The RNA-pol stops moving on the DNA template. The RNA transcript falls off from the transcription complex. • The termination occurs in either  - dependent or  -independent manner.

The  factor, a hexamer, is a ATPase and a helicase.

Model for action of rho factor -independent termination  hexamer binds to protein-free RNA and moves along it. -dependent site  

 '  • The termination signal is a stretch of RNA polymerase transcribes along the template, and  moves along the RNA. 30-40 nucleotides on the RNA transcript, consisting of many GC RNA polymerase pauses at the -dependent terminator site, followed by a series of U. and  catches up • The sequence specificity of this Structure in RNA that causes pausing nascent RNA transcript will form

unwinds the RNA-DNA hybrid particular stem-loop structures to and transcription terminates terminate the transcription. 21/10/62

Stem-loop disruption §2.2 Transcription of Eukaryotes

• The stem-loop structure alters a. Initiation the conformation of RNA-pol, leading to the pause of the • Transcription initiation needs RNA-pol moving promoter and upstream regulatory • Then the competition of the regions. RNA-RNA hybrid and the DNA- DNA hybrid reduces the DNA- • The cis-acting elements are the RNA hybrid stability, and causes the transcription specific sequences on the DNA complex dissociated template that regulate the • Among all the base pairings, the transcription of one or more genes. most unstable one is rU:dA

cis-acting elements for transcription What is TFIID?

• TFIID is TBP with 8-10 TBP associated factors II

• BRE:TFIIB binding element • TATA box • INR: Initiator element • DCE: Down stream core element • DPE: Down stream promoter element 21/10/62

Pre-initiation complex (PIC) Transcriptional process • Pol II Promoter sequence elements include TATA box Resembles –10 sequence element of bacterial promoters • Minimum of 5 GTFs required for in vitro initiation of transcription TFIID (TBP + TAFs), TFIIB, TFIIF, RNAP, TFIIE, TFIIH,

• Additional factors required to initiate transcription: Mediator, large protein complex of more than 20 subunits interacts with both GTFs and pol II

• CTD of pol II is phosphorylated, binds other factors that assist processing, elongation, termination

Transcriptional process: RNAP proximal promoter pause elongation and RNA processing 21/10/62

Chromatin and nonchromatin RNA‐sequencing immunoprecipitation‐sequencing

TF/ChIP‐sequencing ChIP‐sequencing: markers for cis‐elements 21/10/62

b. Elongation c. Termination

• The termination sequence is AATAAA followed by GT repeats. • The termination is closely related to the post-transcriptional modification.

Termination of Eukaryotic RNA Pol II 21/10/62

Modification of hnRNA

Section 3

Post-Transcriptional Modification

§3.1 Modification of hnRNA Capping of pre‐mRNAs

• Primary transcripts of mRNA are called as • Cap=modified guanine nucleotide heteronuclear RNA (hnRNA). • Capping= first mRNA processing event ‐ occurs during transcription • hnRNA are larger than matured mRNA by • CTD recruits capping enzyme as soon as it is phosphorylated many folds • Pre‐mRNA modified with 7‐methyl‐guanosine triphosphate (cap) when RNA is only 25‐30 bp long • Modification includes • Cap structure is recognized by CBC（cap‐binding complex） – Capping at the 5- end • Stablize the transcript – Tailing at the 3-end • Prevent degradation by exonucleases • Stimulate splicing and processing – mRNA splicing 21/10/62

Capping of the 5’ end of nascent RNA transcripts with m7G b. Poly-A tailing at 3 -end

• The cap is added • At 3′ end, poly‐A tail is added by polyadenylation. after the nascent RNA • Signals for cleavage, polyadenylation include: molecules produced • conserved hexanucleotide (AAUAAA in mammalian cells), by RNA polymerase II reach a length of 25- • G‐U rich downstream sequence element. Existing in 30 nucleotides. • Pol II not recognize termination sequences: a single Guanylyltransferase is Fig. 7.46 complex • stops after mRNA cleaved. 20‐40nt recruited and activated through binding to the Ser5-phosphorylated 10‐30nt Pol II CTD.

• The methyl groups are derived from S- Sometimes methylated adenosylmethionine.

• Capping helps stabilize mRNA and enhances translation, Sometimes methylated splicing and export into the cytoplasm.

Consensus sequence for 3’ process Functions of 5’ cap and 3’ polyA

• Need 5’ cap for efficient translation: – Eukaryotic translation initiation factor 4 (eIF4) recognizes and binds to the cap as part of initiation. • Both cap and polyA contribute to stability of mRNA: – Most mRNAs without a cap or polyA are degraded rapidly. – Shortening of the polyA tail and decapping are part of one pathway for RNA degradation in yeast.

AAUAAA: CstF (cleavage stimulation factor F) GU‐rich sequence: CPSF (cleavage and polyadenylation specificity factor) 21/10/62

c. mRNA splicing

Exons are similar in size mRNA

DNA Introns are highly variable in size The matured mRNAs are much shorter than the DNA templates.

Pre-mRNA splicing Pre-mRNA splicing Spliceosome assembly: **Splicing: Introns (noncoding) removed from pre-mRNA 1. U1 snRNP binds to 5′ SS (complementary base pairs) Splicing proceeds in 2 steps: (lariat intermediate) 2. U2 snRNP binds to branch point 3. Other snRNPs join complex, form intron loop • 1. Cleavage at 5′ splice site (SS), joining 5′ end of intron to A within 4. Maintain association of 5′ and 3′ exons: so can be ligated intron (branch point). Intron forms loop. followed by excision of intron. • 2. Cleavage at 3′ splice site, ligation of exons, excision intron

Classical splice site sequences: • 5’ splice site • 3’ splice site • Branch point Fig. 7.47 Fig. 7.49 21/10/62

Pre-mRNA splicing The spliceosome cycle Other splicing factors bind specific RNA sequences and recruit U1 and U2 snRNPs to appropriate sites • SR factors bind specific sequences in exons, • recruit U1 snRNP to 5′ SS. • U2AF binds pyrimidine-rich sequences at 3 ′ SS • recruits U2 snRNP to branch point.

Fig. 7.52

RNA Processing and Turnover RNA Processing and Turnover Aberrant mRNAs can be degraded. Eukaryotic mRNA half-lives vary; < 30 min to 20 hrs • Short-lived mRNAs encode regulatory proteins: Nonsense-mediated mRNA decay eliminates mRNAs levels can vary rapidly in response to environmental stimuli. that lack complete open-reading frames. • mRNAs encoding structural proteins or central • Ribosomes encounter premature termination codons, metabolic enzymes longer half-lives translation stops, defective mRNA degraded • Degradation of mRNAs initiated by shortening poly-A tails. • Ultimately, RNAs degraded in cytoplasm. • Rate of degradation controls gene expression

rRNAs and tRNAs very stable, (both prokaryotes, eukaryotes) • high levels of these RNAs (greater than 90% of all RNA)

Bacterial mRNAs rapidly degraded: t1/2 2 to 3 minutes. • Rapid turnover lets cell respond quickly to changes in environment, such as nutrient availability . Fig. 7.56 21/10/62

mRNA Half‐life The spliceosome cycle

• t½ seconds if seldom needed • t½ several cell generations (i.e. ~48‐72 h) for houskeeping gene • ≈avg 3 h in eukaryotes • ≈avg 1.5 min in bacteria

Errors produced by mistakes The Significance of Gene Splicing in splice‐site selection

• The introns are rare in prokaryotic structural genes • The introns are uncommon in lower eukaryote (yeast), 239 introns in ~6000 genes, only one intron / polypeptide • The introns are abundant in higher eukaryotes (lacking introns are histons and interferons) • Unexpressed sequences constitute ~80% of a typical vertebrate structural gene 21/10/62

Alternative splicing Regulated alternative splicing • Alternative splicing occurs in all metazoa and is especially prevalent in vertebrate Different signals in the pre‐mRNA and different proteins cause spliceosomes to form in particular positions to give alternative splicing

Five ways to splice an RNA

Alternative splicing can generate mRNAs encoding proteins with different, even opposite functions Fas ligand Fas 5 6 7 (membrane- associated)

Fas pre-mRNA (+)

(programmed 576 APOPTOSIS cell death)

(-)

Fas ligand 5 7 Soluble Fas

(membrane)