HSS2305A 2024 Lecture 8 PDF

HSS2305: Molecular Mechanisms of Disease Lecture 7 continued – Interactions Between Cells and Environment; Lecture 8 – Gene Transcription and Translation – Eukaryote Gene Transcription and Translation From Genes to Proteins (Central Dogma of Molecular Biology) From Genes to Proteins (...

HSS2305: Molecular Mechanisms of Disease Lecture 7 continued – Interactions Between Cells and Environment; Lecture 8 – Gene Transcription and Translation – Eukaryote Gene Transcription and Translation From Genes to Proteins (Central Dogma of Molecular Biology) From Genes to Proteins (Complex) From Genes to Proteins http://youtu.be/erOP76_qLWA  From Genes to Proteins Transcription Synthesis of complementary RNA from a DNA template in the nucleus Starting Material: DNA Required machinery: RNA polymerase II Transcription factors End product: messenger RNA (mRNA) after processing Translation Synthesis of proteins in the cytoplasm using information encoded by mRNA Starting Material: mRNA Required machinery: Ribosomal RNA (rRNA) and ribosomal proteins Transfer RNA (tRNAs) End product: polypeptide Sense vs antisense DNA strands -Sense strand -Antisense strand The coding strand’s sequence is identical to the newly synthesized RNA strand, except that thymine (T) in DNA is replaced by uracil (U) in RNA Transcription Both strands encode genes  Transcription RNA Polymerase 5' untranslated region (5' UTR ATG start codon Pre-Initiation Complex Transcription Start Site (+1) Transcription RNA Polymerase https://www.youtube.com/watch?v=JtwMBD7tSGg  Transcription RNA Polymerase RNA polymerase Enzyme that: 1. Binds to DNA 2. Initiates Transcription at a specific site (promoter region) 3. Incorporates nucleotides into a strand of RNA whose sequence is complementary to one of the DNA strands (template) Prokaryotes: One RNA polymerase for transcription of all types of RNA Eukaryotes: Three slightly different RNA polymerases: RNA polymerase I, RNA polymerase II, and RNA polymerase III  Eukaryote Transcription RNA Polymerase RNA polymerase Also knows as the DNA-dependent RNA polymerase RNA is synthesized in the 5’-to-3’ direction The 3’-to-5’ DNA strand that is read to make the RNA strand is called the template strand. start site (+1)  Eukaryote Transcription RNA Polymerase Transcription Factors Proteins that combine with RNA Pol II at the promoter site to form a pre- initiation complex initiate transcription as soon as nucleotides are available Regulate gene expression There are general and specific transcription factors General Transcription Factors/Basal Transcription Factors (GTFs): They assemble at the core promoter and are necessary for RNA polymerase II to begin transcription Consists of TFII A, B, D, E, F, H TF stands for transcription factor and II is for RNA polymerase II TFIID uniquely consists of : TATA-binding protein (TBP) TBP-Associated Factors (TAFs) TFIID’s role is to recognize and bind to the promoter region, initiating the assembly of other transcription factors and RNA polymerase II for transcription. Eukaryote Transcription Gene Promoter Promoter region Site on DNA to which RNA polymerase binds prior to initiation of transcription Determines which strand is template (anti-sense – which becomes sense as mRNA) 100–1000 base pairs long Has specific sequences called elements Core and proximal promoter elements Found within 250 base pairs of the start site Note that most promoters will also have upstream control elements, beyond the core, to improve efficiency Core promoter about -40 to +40 base pairs relative to the TSS Proximal Promoter found upstream of the core promoter, often from about - 250 to -40 base pairs relative to the TSS (note that locations indicated as negative numbers (i.e. upstream of transcription start site – TSS))  Eukaryote Transcription Core Promoter It contains key elements such as the TATA box, initiator sequences, and other motifs essential for the binding of general transcription factors like TFIID and RNA polymerase II. The core promoter is responsible for the basic transcriptional activity of a gene TATA element/box Consensus sequence: TATAWAWR Site of preinitiation complex formation First step in transcription initiation Present at only ∼20% of eukaryotic promoters First eukaryotic core promoter motif to be identified in 1978 Recognized by transcription factor TATA-binding protein (TBP) Possibility for TBP binding to TATA-less promoters B recognition element (BRE) In some human promoters, the TATA box is flanked by BRE Recognized by transcription factor TFIIB Both BREs work in conjunction with the TATA box (and TBP) to have various effects on levels of transcription Note: Consensus Sequence Code (ie TATAWAWR of the TATA box): Y = C or T; W = A or T; R = A or G; N = any base (adenine, thymine, guanine, and cytosine)  Eukaryote Transcription Core Promoter Elements Initiator element (INR) For mammalian RNA polymerase II Overlaps with transcription start site Consensus Sequence: YYANWYY - underlined A indicates start site/+1 Multiple Inr consensus sequences Range from a very relaxed element to a very strict element Recognized by TBP-Associated Factors (TAFs) Downstream promoter element (DPE) Consensus Sequence: RGWYV(T) Role in the initiation of gene transcription by RNA polymerase II Located about 28–32 nucleotides downstream of the transcription start site Recognized by TBP-Associated Factors (TAFs) Eukaryote Transcription Proximal Promoter Elements It includes binding sites for specific transcription factors that regulate the level and timing of gene expression. The proximal promoter enhances the transcriptional activity provided by the core promoter and can influence how frequently transcription initiates https://www.pharmacy180.com/article/transcription-of-eukaryotic-genes-2026/ Important elements commonly found within the proximal promoter include: CAAT Box: A sequence that helps recruit transcription factors to the promoter, enhancing the binding efficiency of RNA polymerase II. GC Box: The GC-rich sequences are binding sites for SP1 and similar TFs. SP1 is involved in activating a broad range of genes, especially those that are constitutively expressed, such as housekeeping genes. Specific Response Elements: These can include sequences responsive to hormones, growth factors, or other signaling molecules, which allow for the regulation of gene expression in response to environmental or cellular signals. Examples: HRE (Hormone Response Element): Binds hormone receptors like the glucocorticoid receptor to regulate genes in response to hormones. CRE (cAMP Response Element): Bound by CREB (cAMP response element-binding protein), allowing regulation in response to cAMP levels. Eukaryote Transcription Enhancer Regions Enhancer region – from 50 to 1500 base pairs long Unlike promoters, enhancers operate at up to 50,000 base pairs upstream of the start site TFs bind to them and with coactivators interact with the promoter region of the target gene to enhance its transcription by binding to the pre-initiation complex. Eukaryote Transcription Pre-Initiation Complex (PIC) Pre-Initiation Complex (PIC): Complex of approximately 100 proteins Necessary for the transcription of protein- coding genes in eukaryotes Position RNA polymerase II at gene transcription start sites Denatures the DNA Positions the DNA in the RNA polymerase II active site for transcription Transcriptional activators (aka specific TFs) bind to coactivators which then bind to the PIC  Eukaryote Transcription Formation of the Pre-Initiation Complex (PIC) https://www.youtube.com/watch?v=EMDuf_kBJcs 1) 1) TFIID (TBP + TAFs) binds to TATA box found upstream of the transcription start site (TSS) in the core promoter (although sometimes found in the proximal 2) promoter) 2) Binding of TFIIA and TFIIB to complex TFIIA stabilizes PIC TFIIB provides specific binding site for RNA Pol II  Eukaryote Transcription Pre-Initiation Complex (PIC) 3) The RNA Pol II:TFIIF complex binds to TFIIB in the PIC 3) TFIIF plays a role in elongation 4) TFIIE and TFIIH bind to the PIC TFIIE helps with binding TFIIH TFIIH contains 3 enzymatic subunits Kinase activity → Phosphorylates 4) sites on RNA Pol II tail to start elongation 2 DNA Helicases → Unwind DNA at promoter start site These all use ATP to form the transcription bubble (13 bp) → open formation Eukaryote Transcription Initiation Transcription initiation: Defined as the formation of a phosphodiester bond between the first two nucleotides of the RNA transcript. TFIIB, TFIIE, TFIIH RNA Polymerase continues to slide forward along the DNA template elongating the RNA Transcription factors of the initiation complex are no longer associated with it Eukaryote Transcription Initiation Carboxyl-terminal domain (CTD) of RNA Polymerase II: 7 aa repeating domain of RNA Pol II subunit Tyr-Ser*-Pro-Thr-Ser*-Pro-Ser (normally polar, uncharged amino acids) TFIIB, TFIIE, TFIIH Serine-5 phosphorylated by the kinase TFIIH CTD becomes more polar and therefore more hydrophilic triggers uncoupling of RNA Pol II from PIC promotes elongation promotes 5` capping of mRNA TFIID remains bound to TATA and can initiate formation of additional PIC  Biological Molecules Proteins R groups weakly acidic or basic not fully charged at pH 7 can form H bonds with other molecules (i.e. H2O) since they have atoms with a partial negative or positive charge  Steps of Eukaryote Transcription Elongation Elongation Extension of mRNA transcript using DNA template RNA Pol II capable of incorporating ~ 20-50 nucleotides into a growing RNA molecule per second Transcription Bubble Unwound section of DNA of approximately 13 bp regions DNA in front of RNA Pol are unwound, compensatory positive supercoils (ch 10) DNA behind RNA Pol are rewound and negative supercoils are present (ch 10) These conditions are relieved by topoisomerases DNA-RNA hybrid ~8-9 bp, stabilizes the elongation complex Eukaryote Transcription Elongation RNA DNA phosphate New complementary nucleotides are incorporated by RNA Pol II into mRNA in 5’→ 3’ direction sugar (or 3’ to 5’ on the DNA template) Ex. base ATP pairs with the thymine containing nucleotide of the template (H-bonds) 3` OH of the previous nucleotide binds to the 5’ α-phosphate of incoming nucleoside triphosphate Pyrophosphate (PPi) is released and hydrolyzed to 2 inorganic phosphates (Pi) releases a large amount of energy → irreversible Eukaryote Transcription Elongation RNA DNA phosphate New complementary nucleotides are incorporated by RNA Pol II into mRNA in 5’→ 3’ direction sugar (or 3’ to 5’ on the DNA template) Ex. base ATP pairs with the thymine containing nucleotide of the template (H-bonds) 3` OH of the previous nucleotide binds to the 5’ α-phosphate of incoming nucleoside triphosphate Pyrophosphate (PPi) is released and hydrolyzed to 2 inorganic phosphates (Pi) releases a large amount of energy → irreversible Eukaryote Transcription Elongation RNA DNA phosphate New complementary nucleotides are incorporated by RNA Pol II into mRNA in 5’→ 3’ direction sugar (or 3’ to 5’ on the DNA template) Ex. base ATP pairs with the thymine containing nucleotide of the template (H-bonds) 3` OH of the previous nucleotide binds to the 5’ α-phosphate of incoming nucleoside triphosphate Pyrophosphate (PPi) is released and hydrolyzed to 2 inorganic phosphates (Pi) releases a large amount of energy → irreversible Eukaryote Transcription Elongation Elongation Transcription Factors: > 50 components involved in elongation P-TEFb (Positive Transcription Elongation Factor b) phosphorylates the CTD at Ser-2 (Tyr- Ser*-Pro-Thr-Ser*-Pro-Ser) Recruits factors that help elongation or RNA modifications splicing and polyadenylation (discussed later) ELL Eleven-Nineteen Lysine-rich Leukemia protein) and TFIIF weakens interactions between RNA Pol II and nonspecific binding sites of DNA suppressing transient pausing of the polymerase TFIIS stimulates elongation helps get RNA Pol II moving after pauses, proofread and correction of mistaken nucleotides Eukaryote Transcription Termination In prokaryotic bacterial cells Well defined termination sequences In eukaryotic cells No well-defined sequence transcription termination is often associated with the polyadenylation signal sequence (PAS), which typically appears as AAUAAA in the RNA transcript ? Ultimate length of mRNA (3’ end) is determined by processing steps Cleavage of the new transcript is followed by template-independent addition of Adenosines at its new 3' end → polyadenylation AAUAAA sequence well past end of coding region (poly A tail) Questions?