MM Biochemistry BL5 Transcription PDF

MM Biochemistry BIOCHEMISTRY BL5 Transcription and Post-transcriptional modification DR. KOMALA THIRUMALAI  Transcription process of making mRNA from a DNA template  LEARNING OUTCOMES Transcription 1: Events of Transcription Learning Out...

MM Biochemistry BIOCHEMISTRY BL5 Transcription and Post-transcriptional modification DR. KOMALA THIRUMALAI  Transcription process of making mRNA from a DNA template  LEARNING OUTCOMES Transcription 1: Events of Transcription Learning Outcomes: Content: At the end of this session the students would be able to: Define transcription Definition of transcription Description of transcription process in a Description of transcription process sequential manner o Enzymes and substrates involved o Transcription factors o Events of transcription: ▪ Initiation ▪ Elongation ▪ Termination Explain the significance of post Significance of Post-transcriptional processing transcriptional modifications o Removal of introns and splicing o Role of spliceosomes and snRNA Name the antimicrobial agents that inhibit Antimicrobial agents that inhibit transcription of microbial transcription of microbial genome genome o Antibacterial o Antiviral o Antifungal 3  Events in the central dogma The traits or phenotypes are regulated by genes. How does a gene determine / express its phenotype The central dogma refers to a sequential information transformation from DNA → RNA →proteins Two-step process DNA → mRNA → protein 4 Transcription DNA acts as a template for transcription required RNA polymerase Unlike DNA replication, transcription is highly selective and tightly regulated A cellular process to synthesize RNA ○ Coding RNA ○ Non-coding RNA 5  Coding: mRNA (encodes Non-coding: rRNA, tRNA, siRNA, protein to act as various miRNA, snRNA (act as cellular components including enzymes regulators) and cell structures) Doesn’t provide instructions for Provides instruction for making making proteins proteins Determines when and where genes are turned on and off 6 Coding RNAs General function mRNA - DNA template to be translated into a functional protein Non-coding General functions RNAs tRNA (transfer - An adaptor to link mRNA and amino This Photo by Unknown Author is RNA) acids during translation and decode licensed under CC BY genetic information from mRNA rRNA (ribosomal - Involved in ribosome formation which RNA) catalyzes the translation process snRNA - Processing of pre-mRNA in the (small nuclear nucleus RNA) - Splicing pre-mRNA (remove intron) This Photo by Unknown Author is licensed under CC BY 7 Non-coding RNAs General functions miRNA - Regulate gene expression (microRNA) snoRNA - Involved in processing rRNA → rRNA maturation This Photo by Unknown Author is licensed (small nucleolar RNA) under CC BY-SA-NC siRNA - Inhibiting /interfering translation (short interfering RNA) * Potential strategy for treatment of many human diseases lncRNA - Involved in chromatin re-modeling, as well as (long non-coding RNA) transcriptional and post-transcriptional regulation This Photo by Unknown Author is licensed under CC BY-SA- NC 8  Transcription process SIGNAL 1. Initiation complex formation Eukaryotes vs prokaryotes 2. Elongation RNA polymerase Transcription process is well- 3. Termination studied in prokaryotes whereas eukaryotic transcription is more complex Processing 9 Gene Definition ○ Entire deoxyribonucleic acid sequence that is required for a synthesis of a functional polypeptide DNA sequences ○ Coding region Exon ○ Non-coding region Regulatory elements Intron Chorev and Carmel, 2012. The function of introns. Frontiers in genetics, 3 (55), 1 – 15. https://doi.org/10.3389/fgene.2012.00055 10  Regulatory elements Cis-acting elements Trans-acting elements DNA sequence Not DNA sequence Latin – “cis” – “same side as” Regulate the expression of gene by binding to A region of DNA or RNA that regulates the cis-acting DNA sequence expression of genes located on the same Diffuse through cytoplasm and act at the molecule target DNA sites Influence the expression of adjacent genes on Usually refer to protein the same DNA molecule Examples: Examples: i. General transcription factors Core promoters TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH Proximal promoter elements basal level of gene expression Enhancer ii. Gene specific transcription factors Silencer i. Activator ii. Repressors 11 Trans-acting elements TFIID – binds to the sequence TATA TFIIH – facilitates promoter melting * Essential component in nucleotide excision repair ○ Location: (upstream of coding site) The start site of transcription is termed as +1 Upstream bases are designed as negative number No bases are designated as 0 Promoters: A generalized promoter of a gene transcribed by RNA polymerase II is shown. Transcription factors recognize the promoter. RNA polymerase II then binds and forms the transcription initiation complex. TATA-binding protein (TBP) 13 Examples of cis-acting elements Enhancer/Silencer GC GC GC GC TATA Inr Enhancer/Silencer DNA strand +1 (start site of transcription) Cis-acting elements Examples Location on the DNA Core promoter TATA box -31 to -26 Inr: Initiator -2 to +4 Proximal promoter GC box -37 to -250 element Enhancer / Silencer -72bp varies Promoters provide binding sites for the protein necessary for the regulation machinery that carries out transcription. of gene expression 14 RNA polymerase ○ Multi subunits enzyme ○ General functions: Recognizes and binds to the promoter region Synthesize mRNA using DNA template 15  RNA polymerase in prokaryotes σ (sigma) subunit: recognize Core enzyme promoter regions and initiate ○ 4 peptide subunits: 2α, 1β, 1β‘ transcription ○ 2α: enzyme assembly ○ β‘: template binding ○ β: 5'→3' RNA polymerase activity Core enzyme to ensure that RNA polymerase bind to the DNA while the σ factors are responsible for the ability of RNA polymerase to recognize the promoter sequence DNA and initiate transcription. Holoenzyme = combination of an enzyme with a 16 coenzyme RNA polymerase in Eukaryotes All eukaryotes have three different RNA polymerases (RNAPs) which transcribe different types of genes. ○ RNA polymerase I Synthesize large rRNA (28S, 18S and 5.8S) / transcribes rRNA genes ○ RNA polymerase II Synthesize precursor mRNA and snRNAs / transcribes mRNA, miRNA, snRNA and snoRNa genes ○ RNA polymerase III Synthesize tRNA, 5S rRNA and snRNAs / transcribes rRNA and 5S rRNA genes 17 1. Formation of the initiation complex in prokaryotic transcription RNA polymerase recognize and bind to the promoter region at ○ Pribnow box (TATAAT) ○ -35 sequence (TTGACA) Any mutations at these regions ○ Disrupt transcription ○ No gene product The binding of RNA polymerase at the promoter site marks the activation/beginning of DNA transcription 18 1. Formation of the initiation complex in eukaryotic transcription Transcription factors ○ recognize and bind to the promoter region ○ recruit the binding of RNA polymerase ○ formation of Initiation Complex 19 2. Elongation RNA polymerase synthesizes RNA in 5’ → 3’ direction by moving towards the downstream of DNA sequence. Complementary rule is similar with AT/GC rule, except that T is replaced by Uracil (U) In prokaryotes ○ Same RNA polymerase synthesizes all types of RNAs In eukaryotes ○ RNA polymerase I ○ RNA polymerase II ○ RNA polymerase III 20 3. Termination A process to detach newly synthesized RNA strand from DNA template Prokaryotes ○ Rho dependent mechanism ○ Rho independent mechanism Eukaryotes ○ Not well understood ○ But different RNA polymerase require different termination mechanism ○ More complex than prokaryotes But share some similarities 21 3. Termination : Prokaryotes A. Rho-dependent termination: Rho protein ○ Adenosine triphosphatase (ATPase) with helicase activity ○ Unwind RNA-DNA hybrid strands ○ Release the newly synthesized RNA strand. 22 3. Termination : Prokaryotes B. Rho-independent termination Presence of palindrome sequence in DNA ○ Reading sequence is similar in both 5’→3’ and 3’→5’ directions ○ Generate “self-complementary” sequence in RNA 5’ 3’ RNA to fold 3’ 5’ back on itself → “hairpin ” (stabilized by H- bonds and a loop structure) 5’ 3’ 23 3. Termination : Prokaryotes B. Rho-independent termination The newly synthesized RNA is rich in Uracil (U) at the 3’ end U-A bonding is weak ○ RNA detach from DNA 24  Transcription process overview (eukaryotes) Termination Antisense (non-coding DNA strand of a region 3’ gene) 5’ Promoter region Gene ( introns + exons) 5’ RNA 3’ RNA polymerase TFs 1. Initiation 2. 3. Elongation Termination 25  Transcription Prokaryotes Eukaryotes Occur in the cytoplasm Occur in the nucleus RNA polymerase initiate Transcription factors initiate the transcription formation of initiation complex No introns v Presence of introns No splicing for mRNA Pre-mRNA is subjected to splicing Same RNA polymerase synthesize There are 3 RNA polymerase all types of RNAs Monocistronic mRNA (one gene Polycistronic mRNA (can code code per one mRNA) many genes) QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my 26 26 Post-transcriptional modification : mRNA mRNA of prokaryotes not subjected to post transcriptional modification. Translation started simultaneous with transcription tRNA and rRNA of eukaryotes undergo post transcriptional modification 27  mRNA processing: Post-transcriptional modification Primary mRNA processed first and then transported to the cytoplasm 3 main steps in mRNA processing ○ 5’ cap ○ Poly A tail at 3’ end ○ Splicing – to remove junk sequences (introns) occurs in nucleus UTR – untranslated region 28 Post-transcriptional modification : mRNA The first processing is 5’ capping ○ Addition of 7-methylguanosine to the 5’ terminal end of the mRNA ○ Forming 5’→5’ triphosphate linkage ○ Catalyzed by guanylyltransferase ○ Permits the initiation of translation and stabilize the mRNA Lacking the 5’ cap, mRNA cannot be translated. 29 Post-transcriptional modification : mRNA Addition of poly-A tail ○ mRNA possesses a polyadenylation signal sequence at the 3’ end ○ Lead to addition of poly-A tail to the 3’ end of mRNA ○ Stabilize the mRNA ○ Facilitate their exit from the nucleus. ○ After the mRNA enter the cytosol, poly-A tail is gradually shortened. 30 Post-transcriptional modification : mRNA Splicing most of the bacterial mRNA does not undergo splicing ○ Occur in the nucleus ○ Eukaryotic pre-mRNA → mature mRNA ○ Small nuclear RNAs (snRNA) + proteins to form a complex → small nuclear ribonucleoprotein particles (snRNPs) ○ snRNPs facilitates the cleavage of introns ○ Splice sites found in intron 5′ end : GU 3′ end : AG 31 Post-transcriptional modification : rRNA Primary transcript is called preribosomal RNAs ○ In eukaryote, RNA polymerase I & III synthesize rRNAs ○ In prokaryote, rRNA is synthesized by RNA polymerase ○ Cleaved by ribonucleases ○ Generate 28S, 18S and 5.8S (eukaryote) 23S, 16S and 5S (prokaryote) 32 Post-transcriptional modification : tRNA Post-transcriptional enzyme-catalyzed modification of tRNA occurs at several base and sugar positions and influences specific anticodon–codon interactions and regulates translation, its efficiency and fidelity. 33 Transcription inhibitors Rifamycin antibacterial agents – rifampin, rifapentine, rifabutin and rifamixin – bind to and inhibit bacterial RNA polymerase Treatment for gram-positive and gram-negative bacterial infections Binds to β-subunit of RNA polymerase For tuberculosis (not suitable for common cold, flu) Has no effect on eukaryotic nuclear RNA polymerases 34 Transcription inhibitors Actinomycin D / Dactinomycin Transcription inhibitor Binds to DNA template (binds to double helical DNA – prevents it from being an effective template for RNA synthesis) Antibacterial and antitumor activity (anticancer) Shows major regressions (decrease in the size of a tumor) of pancreatic and stomach cancer xenografts* (Liu et al., 2016) *refers to a tissue or organ that is derived from a species that is different from the recipient of the specimen. 35 Transcription inhibitors α-amanitin  As a potent inhibitor of RNA polymerase II, α- amanitin is toxic to eukaryotic cells  Binds to RNA polymerase Amanita → inhibit mRNA synthesis phalloides Inhibition of this transcription or translation step has the effect of blocking protein production and ultimately its function. 36 THANK YOU