TzivionMCB3TranscriptionF2024.pptx

Transcription Dr. Guri Tzivion Office: GC18 [email protected]  Learning Objectives MCB. 3. Understand how genes encoded in DNA are transcribed into messenger RNA for eventual translation into proteins. By the end of this module, students should be able to: MCB.3...

Transcription Dr. Guri Tzivion Office: GC18 [email protected]  Learning Objectives MCB. 3. Understand how genes encoded in DNA are transcribed into messenger RNA for eventual translation into proteins. By the end of this module, students should be able to: MCB.3.1. Describe the process of transcription, with emphasis on gene organization, the direction of transcription, general and specific transcription factors, and the relevant enzymes in prokaryotes and eukaryotes. MCB.3.2. Identify the types of RNA modifications and the role of small nuclear ribonucleoproteins (snRNPs) in the maturation of mRNA during post- transcriptional processing. Textbook Reading: Lippincott's Illustrated Reviews – Biochemistry 8th ed. Chapter 31: RNA Structure, Synthesis, and Processing Read sections: Overview Prokaryotic Gene Transcription Prokaryotic RNA polymerase Steps in RNA synthesis Eukaryotic Gene Transcription Chromatin structure and gene expression Nuclear RNA polymerase Posttranscriptional modification of RNA  DNA is the Genetic Material, therefore it must: (1) Replicate faithfully. (2) Have the coding capacity to generate proteins and other products for all cellular functions. “A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.” Francis Crick  Central dogma replication transcription translation DNA RNA protein reverse transcription Central dogma DNA mRNA Replicatio Transcriptio n n Nucleus + Translation Cell Cytosol Protein The Central Dogma  Transcription is a process in which:  The sequence of bases in a particular stretch of DNA (a gene) specifies the sequence of bases in an mRNA molecule.  Translation is a process in which:  A particular mRNA molecule then specifies the exact sequence of amino acids in a protein.  Thus, genes ultimately code for proteins.  Reverse transcription: synthesis of DNA using RNA as the template  RNA (Ribonucleic acid )  RNA is a polymer of ribonucleotides linked together by 3’-5’ phosphodiester linkage MAJOR CLASSES OF RNA  rRNA (ribosomal RNA)  mRNA (messenger RNA)  tRNA ( transfer RNA)  snRNA (small nuclear RNA) hn RNA (heterogeneous nuclear RNA)  miRNA (micro RNA) DNA: Deoxyribose Nucleic Acid Nitrogenous base (A, G, C, or T) Phosphate group Thymine (T) Sugar (deoxyribose) RNA: Ribose Nucleic Acid Nitrogenous base (A, G, C, or U) Phosphate group Uracil (U) Sugar (ribose)  Similarities 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´  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  What is a Gene ? The fundamental physical and functional unit of heredity. A gene is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product (i.e., a protein or RNA molecule) It is a functional unit that is regulated by transcription and encodes an RNA product, which is most commonly, but not always, translated into a protein that  The whole genome of DNA needs to be replicated, but only a small portion of the genome is transcribed in response to developmental requirements, physiological needs and environmental changes.  DNA regions that can be transcribed into RNA are called structural genes.  Points of control The control of gene expression can occur at any step in the pathway from the gene to a functional protein 1. Packing/unpacking of DNA 2. Transcription 3. mRNA processing 4. mRNA transport 5. mRNA stability 6. Translation 7. Protein processing 8. Protein activity 9. Protein degradation AP Biology What is Gene Expression ???  The process by which a gene's coded information is converted into the structures present and operating in the cell  Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (e.g., transfer and ribosomal RNAs)  The activity of a gene can be determined by examining the production of the RNA or the Transcription in Bacteria  In transcription:  Instructions stored in DNA are “transcribed” through the synthesis of an mRNA transcript.  Transcription performed by RNA polymerase.  Three phases of transcription:  Initiation, elongation, and termination.  Transcription in Bacteria  mRNA transcript is synthesized by RNA polymerase in the 5’ to 3’ direction.  Template strand:  DNA strand that is read (transcribed) by RNA polymerase.  Non-template strand:  Template The template strand is the strand from which the RNA is actually transcribed. It is also termed as antisense strand. The coding strand is the strand whose base sequence specifies the amino acid sequence of the encoded protein or the sequence of the RNA transcript. Therefore, it is also called the sense strand. Transcription: coding and template strands  Transcription in Bacteria “Activated”(tri-phosphate) complementary ribonucleotide monomers are added on via condensation reactions, resulting in phosphodiester bonds. Bacterial RNA polymerase The bacterial RNA polymerase holoenzyme is made up of a core enzyme, consisting of 4 subunits that synthesize the mRNA, and of a regulatory subunit, sigma (s), which coordinates transcription initiation.  The RNA polymerase of E. Coli subunit MW function Identifies the DNA region to  36512 be transcribed  150618 Catalyzes the polymerization Binds and opens the DNA   155613 template Recognizes the promoter  70263 region for synthesis initiation Phase 1: Transcription initiation  Transcription initiation begins at specific sequences of DNA called promoters.  Two important bacterial promoter regions :  Named the -10 box and the -35 box.  The sigma subunit recognizes these promoter regions and brings the RNA polymerase core enzyme to the promoter to initiate transcription.  Transcription initiation 1. Sigma binds to specific promoter regions of DNA (-35 box and -10 box) Promoter site: -35 nucleotides – TTGACA – the initial point of contact for the holoenzyme -10 Pribnow box (TATA box) - TATAAT Mutations in either the -35 box or the Pribnow (TATA) box can affect the rate of transcription  Transcription initiation 2. The DNA double helix is opened, and the template strand of DNA is threaded through the RNA polymerase core enzyme active site: mRNA synthesis begins.  Transcription initiation 3. Initiation is complete: Sigma dissociates from the core enzyme and mRNA synthesis (transcription) continues. Transcription initiation  Phase 2: Transcription elongation  The core enzyme of RNA polymerase moves along the DNA template and continues to catalyze the addition of complementary ribonucleotides to the growing mRNA transcript. Phase 3: Transcription termination RNA polymerase encounters a termination signal within the DNA template, which codes for a hairpin loop structure in the RNA. The hairpin causes the RNA polymerase to separate from the RNA transcript, ending the transcription. Phase 3: Transcription termination RNA polymerase encounters a termination signal within the DNA template, which codes for a hairpin loop structure in the RNA. The hairpin causes the RNA polymerase to separate from the RNA transcript, ending the transcription.  Bacterial Transcription: Summary  Initiation:  Sigma brings the RNA polymerase holoenzyme to the promoter region of DNA.  DNA helix is opened and transcription begins.  Sigma is released and transcription continues.  Elongation:  Complementary ribonucleotides are added to the growing mRNA transcript as specified by the DNA template strand.  Termination:  RNA polymerase reaches a termination signal in the DNA template.  mRNA forms a hairpin loop. Clinical Significance Antibiotic Inhibitors of Transcription Rifampin binds to prokaryotic Rifampin (Rifampicin) RNA pol & Binds to the β subunit of prokaryotic prevents chain RNA polymerase extension beyond 3 nucleotides  Transcription in Eukaryotes Overall similar to bacterial transcription. Some differences include: Basal transcription factors: Proteins that bind DNA promoters independent of RNA polymerase. RNA polymerase then binds to basal transcription factors and transcription begins. Greater diversity and complexity of promoters: Many promoters include a TATA box sequence. Eukaryotic Transcription Nuclear Cytoplasm pores DNA Transcription RNA RNA Processing mRNA G AAAAAA G AAAAAA Export Nucleus  Transcription in Eukaryotes Three types of nuclear RNA polymerases: RNA polymerase II: Catalyzes transcription of genes that code for proteins, forming mRNA. RNA polymerase I and RNA polymerase III: Catalyzetranscription of non-protein coding genes (e.g. genes coding for ribosomal rRNAs and genes coding for transfer tRNAs respectively). Requirements for eukaryotic RNA transcription Enzymes - 3 types of nuclear RNA polymerases: - RNA polymerase I: - Synthesizes precursors of 28S, 18S and 5.8S rRNA - RNA polymerase II: - Synthesizes precursors of mRNA - Synthesizes non-coding RNAs - small nuclear RNA (snRNA) - microRNA (miRNA) - RNA polymerase III: - Synthesizes precursors of tRNA - Synthesizes 5S rRNA and some snRNA - Mitochondrial RNA polymerase - Transcription Factors Requirements - DNA template - Euchromatin: Transcriptionally active - Heterochromatin: Transcriptionally inactive (Constitutive/Facultative) Chromatin remodeling (Epigenetic Programming) - Histone modification - DNA methylation (hyper methylation) - Substrates: - Ribonucleotides Ribonucleoside Tri Phosphates (rNTPs)  Requirements (contd..) - Transcription Factors (TFs): - General term for any protein, other than RNA polymerase, required to initiate or regulate transcription in eukaryotic cells. - Defined as trans-acting factors - Examples: SL1 for RNA pol I, TFIID for RNA pol II, TFIIIC for RNA pol III - They bind to DNA through a variety of DNA binding domains - They may bind to the core promoter site, close proximity elements or distal elemnets - They are required for the assembly of the transcription initiation complex at the promoter region and the determination of which genes are to be transcribed  - Transcription Factors (TFs) (contd..): - Two types - General TFs - Required for transcription of all genes, participate in formation of the transcription-initiation complex near the transcription initiation site and therefore constitute basic transcription machinery - Transcription Factors (TFs) (contd..): - Specific TFs - Stimulate (or repress) transcription of particular genes by binding to their regulatory sequences. - Also called as Trans-acting factors/ Transcriptional activators - Required to modulate the frequency of initiation - Required to mediate the response to signals (hormones) through binding to promoter and enhancer elements - Required to regulate which genes are expressed at a given point in time Examples: Sp1, CTF, c-Myc, c-jun, Glucocorticoid receptor  Transcription Factors Core Promotor elements are bound by General Transcription Factors. Additional consensus sequences lie upstream of the core promoter: those close to the core promoter, within ~200 nucleotides, are defined as proximal regulatory elements, such as the CAAT and GC boxes. Those farther away are the distal regulatory elements such as enhancers. Specific transcription factors, also called transactivators, bind regulatory elements outside of the core promotor Transcription factors involved in eukaryotic transcription 46 Transcription initiation in eukaryotes: - RNA pol II cannot bind to the promoter site on its own - TFIID identifies and binds to the promoter site - TFIIF recruits RNA pol II - TFIIH by its helicase activity unwinds DNA molecule and transcription starts Each gene is transcribed from its own promoter, producing one mRNA, which generally is translated to yield a single polypeptide Transcription initiation in eukaryotes: For certain genes, enhancer elements recruit specific transcription factors for promoting transcription initiation Clinical Significance: α-amanitin, a toxic compound in the poisonous mushroom Amanita phalloides (“Death Cap”) inhibits RNA polymerase II Actinomycin D inhibits transcription by binding and intercalating DNA – used in cancer treatment MCB. 3. Understand how genes encoded in DNA are transcribed into messenger RNA for eventual translation into proteins. By the end of this module, students should be able to: MCB.3.1. Describe the process of transcription, with emphasis on gene organization, the direction of transcription, general and specific transcription factors, and the relevant enzymes in prokaryotes and eukaryotes. MCB.3.2. Identify the types of RNA modifications and the role of small nuclear ribonucleoproteins (snRNPs) in the maturation of mRNA during post- transcriptional processing. Eukaryotic Transcription Nuclear Cytoplasm pores DNA Transcription RNA RNA Processing mRNA G AAAAAA G AAAAAA Export Nucleus Eukaryotic mRNA processing - No modification in prokaryotes - Co- and post-transcriptional modifications in eukaryotes - Pre mRNA is generally referred to as heterogeneous nuclear RNA (hnRNA) gene hnRNA - 5’ Capping - Addition of Poly-A-tail - Removal of introns & ligation of exons (Splicing) mRNA  Eukaryotic mRNA Processing: 5’ capping and poly(A) tailing Besides splicing, other steps involved in mRNA processing include:  Addition of a “cap” at the 5’ end of the mRNA. The “5’ cap” serves as a recognition signal for the translation machinery of the cell.  Addition of poly (A) tail at the 3’ end of the mRNA. Serves to stabilize the mRNA by protecting it from enzymatic degradation. mRNA processing - 5’ Capping: - Takes place at the 5’ end of the RNA shortly after transcription initiation - involves addition of 7-methyl GTP at the 5’ end - Enzymes: 1. Guanyl transferase - Adds GTP to the 5’ end as 5’- 5’ tri phosphate linkage 2. Guanine methyl transferase - Adds methyl group to Guanine residue at 7th position Functions: - Stabilizes mRNA - Serves as ribosome-binding site - mRNAs lacking the Cap are not efficiently translated Messenger RNA (mRNA) PTM (contd..) - Addition of poly-A-tail at 3’ end: - Addition of about 200-250 “A” residues at 3’ end - Signal: - Hexonucleotide AAUAAA at the 3’ end (in addition to upstream and down stream (GU rich elements) - mRNA is cleaved downstream of the signal, followed by polyadenylation - Enzyme: - Polyadenylate polymerase (uses ATP as substrate) Function: - Stabilizes mRNA - Facilitates exit of mRNA from the nucleus - Helps in translation - mRNAs lacking Poly-A-tail are susceptible to degradation  Eukaryotic mRNA Processing: splicing Exon 1 Exon 2 Exon 3  After transcription, specific regions of the primary RNA transcript are spliced out and degraded during RNA processing.  Exon: Contains sequences required for protein synthesis and is NOT spliced out during RNA processing. Is part of the final mature spliced RNA transcript.  Intron: Spliced out during RNA processing and degraded. Eukaryotic mRNA splicing Intron regions of the primary mRNA transcript are spliced out by small nuclear ribonucleoproteins (snRNPs), which assemble to form a spliceosome (a ribozyme). This process occurs in the nucleus. Eukaryotic mRNA splicing Eukaryotic mRNA splicing Splicing process: Note the location of the splice donor and acceptor sites 60 Ribosomal RNA (rRNA) processing Both in prokaryotes and eukaryotes Transfer RNA (tRNA) processing Clinical Significance of PTM - About 15% of all genetic diseases are a result of mutation affecting splicing !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! - β-thalassemia - β-globin gene of Hb is severely under expressed - Results from a nucleotide change at an exon-intron junction, precluding removal of the intron and therefore leading to diminished or absent synthesis of the β-globin chain protein - Presents with hemolytic anemia - Systemic lupus erythematosus (SLE): - Autoimmune disease - Antibodies against host proteins eg: snRNP - Fatal - Gaucher disease - Tay-Sach’s disease  Transcription: Summary  Note the similarities and differences between bacterial vs. eukaryotic transcription! Prokaryote versus Eukaryote Transcription Questions? Break