Module 3 Molecular Biology PDF

MIT School of Bioengineering Sciences and Research (A Constituent unit of MIT ADT University) Module: 3 Module Name: Transcription and post transcriptional modifications Course Code: 21BBT3...

MIT School of Bioengineering Sciences and Research (A Constituent unit of MIT ADT University) Module: 3 Module Name: Transcription and post transcriptional modifications Course Code: 21BBT301 Course Coordinator: Course Name: Molecular Biology Contact Number: Module: 3 Mail ID: Disclaimer: The content delivered here should be considered of utmost importance. However, it is to be noted that, this material is not Stand-alone material for the fulfilment of the course syllabus. The content in this presentation should only be used as an aid to learning. Books and other resources provided are suggested to be referred for exhaustive understanding. MITBIO/MITADT University Objective/Learning Outcome: Understand the principles behind the central dogma of molecular biology: DNA replication, transcription, and translation. Enable to learn the regulation of gene expression in prokaryotes and Eukaryotes. MITBIO/MITADT University Syllabus: Fine structure of prokaryotic and eukaryotic gene, structure and function of the promoters in mRNA, rRNA, tRNA genes. RNA polymerases in prokaryote and eukaryote, types and function. Transcription of mRNA, rRNA, and tRNA genes in Prokaryotes and Eukaryotes. Post transcriptional processing of mRNA – 5’capping, splicing, polyadenylation and RNA editing. MITBIO/MITADT University  Gene ○ Part of DNA, where the particular DNA sequence determines the function. Genome ○ the haploid set of chromosomes in a gamete or microorganism, or in each cell of a multicellular organism. ○ the complete set of genes or genetic material present in a cell or organism. Open reading frames (ORFs) ○ spans of DNA sequence between the start and stop codons Operon ○ A functioning unit of DNA containing a cluster of genes under the control of a single promoter Riboswitch ○ In molecular biology, a riboswitch is a regulatory segment of a messenger RNA molecule that binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA transcription factor (TF) ○ In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence Source WikiJournal of Medicine, 2017, 4(1):2 Figure Article, available at: https://doi.org/10.15347/wjm/2017.002 MITBIO/MITADT University  enhancer ○ A short (50–1500 bp) region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur Repressor ○ DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A transcriptional activator ○ Protein (transcription factor) that increases transcription of a gene or set of genes silencer ○ In genetics, a silencer is a DNA sequence capable of binding transcription regulation factors, called repressors. Promoter ○ a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. A regulatory sequence ○ a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of specific genes within an organism. Source WikiJournal of Medicine, 2017, 4(1):2 Figure Article, available at: https://doi.org/10.15347/wjm/2017.002 MITBIO/MITADT University  Features of prokaryotic genome ○ Most prokaryotic cells are small and most genes are expressed at relatively low levels. Cell doubles in 20 min (fast). DNA interacts directly with molecular machines in the cytoplasm. ○ size of prokaryotic genomes ranges from around 50 kb to more than 13 Mb ○ genomes are also very compact, with gene density typically approaching 85% Shortcomings of prokaryotic genome ○ smaller genomes are nearly depleted of sensory, transport, communication, and regulatory functions, reflecting narrow environmental ranges ○ Natural selection is more efficient in larger genomes, possibly as a result of larger effective population sizes. ○ Larger genomes correspond to more versatile prokaryotes that are less sexually isolated and in which selection is more efficient. Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University  Fine structure of prokaryotic genome ○ lack a clear physical separation between DNA and the cytoplasm. cell doubles in 20 min. DNA interacts directly with molecular machines in the cytoplasm. ○ high rates of horizontal gene transfer and gene loss. ○ Genome replication, segregation, and cell doubling are tightly linked ○ Transcription, translation, and protein localization are tightly linked & nascent transcripts are immediately translated by multiple ribosomes. ○ cells endure intense gene expression and collisions between the rapid replication fork and the relatively slower RNA polymerases are frequent. ○ SOS or SOS-like responses may kick off the transfer of mobile genetic elements to other cells Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University  Traits associated with polyploidy in prokaryotes. The presence of multiple copies of replicons, and particularly chromosomes, has been proposed to confer several advantages. (A) It allows distributing gene expression through the entire cytoplasm in very large cells. It allows heterozygosity. In the presence of several replicons, the ratio between replicons allows gene expression regulation. (B) It allows repair by homologous recombination with other similar replicons. (C) Recombination between similar replicons also allows gene conversion and allelic exchange. Heterozygosity is indicated using distinct colors (red and black). Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University  Organization of prokaryotic genome ○ Prokaryotes endure high rates of rearrangement, mutation, deletion, and accretion of genetic material. ○ Genomes are polyploid, with certain species carrying more than 100 copies of the chromosome per cell, allowing distributing gene expression through the entire cytoplasm in very large cells. (heterozygosity). ○ In the presence of several replicons, the ratio between replicons allows gene expression regulation.Facilitates repair and homologous recombination ○ In newly replicated cells, the macrodomains around the origin (Ori) and terminus (Ter) of replication are localized near opposite cell poles leading to a linear arrangement of the genetic information in the cell ○ Thus, completely symmetric, with origin and terminus separated by ∼180° in circular replicons, so that both forks complete replication synchronously. multiple replication forks are common in fast growing bacteria Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University  Elements of Prokaryotic genome organization Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University MITBIO/MITADT University  Operons in prokaryotic genome ○ The majority of genes in prokaryotes are expressed under the form of polycistronic units called operons (may contain 2-12; average ∼3–4 genes: often encode physically interacting proteins, often encode enzymes of consecutive steps in metabolic pathways) ○ Organization of genes in operons is a compact way of regulating gene expression because genes in the same operon are expressed at more similar rates than random pairs of genes ○ SAFER: Pairs of contiguous genes in operons are highly conserved showing rearrangement rates orders of magnitude lower than other interoperonic pairs ○ Large genomes have more complex genetic networks and many more different transcription factors than small genomes. Thus, there is increased selection for operons in small genomes. Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University  The genetic organization of gene expression Schematic representation of the transcription factory model, in which multiple active RNA polymerases are concentrated at discrete sites in the nucleoid Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University  The genetic organization of prokaryotic gene expression (B) Bacterial genes are organized into operons, which group in superoperons. In addition to being physically close in the genome, genes in operons are cotranscribed, coregulated, and encode proteins involved in the same functional pathway or protein complex. Superoperons are not cotranscribed, but may share regulatory regions. Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University  The genetic organization of prokaryotic gene expression (C) Schematic representation of three models aiming at explaining the formation and conservation of operons based on genetic linkage. Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University  The genetic organization of prokaryotic gene expression (D) Transcription of genes in a single transcript is expected to diminish gene expression noise and ensure more precise stoichiometry. It also allows responding optimally to demand for a given pathway when the first genes to be transcribed are those starting the functional pathway. (E) Operons place several genes under the same regulatory region that is thus subject to more efficient selection. Source: Touchon M, Rocha EP. Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harb Perspect Biol. 2016 Jan 4;8(1):a018168. doi: 10.1101/cshperspect.a018168. PMID: 26729648; PMCID: PMC4691797. MITBIO/MITADT University  Prokaryotic Gene structure ○ ORFs are often grouped into a polycistronic operon under the control of a shared set of regulatory sequences.These ORFs are all transcribed onto the same mRNA and so are co-regulated and often serve related functions. ○ Each ORF typically has its own ribosome binding site (RBS) so that ribosomes simultaneously translate ORFs on the same mRNA ○ Having multiple ORFs on a single mRNA is only possible in prokaryotes because their transcription and translation take place at the same time and in the same subcellular location. ○ The operator sequence next to the promoter is the main regulatory element in prokaryotes. Repressor proteins bound to the operator sequence physically obstructs the RNA polymerase enzyme, preventing transcription ○ Riboswitches switch between alternative secondary structures in the RNA depending on the concentration of key metabolites. The secondary structures then either block or reveal important sequence regions such as RBSs. ○ Introns are extremely rare in prokaryotes and therefore do not play a significant role in prokaryotic gene regulation. Source: WikiJournal of Medicine, 2017, 4(1), Figure Article. Available at: https://doi.org/10.15347/wjm/2017.002 MITBIO/MITADT University  The structure of a prokaryotic operon of protein-coding genes. Regulatory sequence controls when expression occurs for the multiple protein coding regions (red). Promoter, operator and enhancer regions (yellow) regulate the transcription of the gene into an mRNA. The mRNA untranslated regions (blue) regulate translation into the final protein products MITBIO/MITADT University  Eukaryotic Gene structure ○ ORFs are often grouped into a polycistronic operon under the control of a shared set of regulatory sequences.These ORFs are all transcribed onto the same mRNA and so are co-regulated and often serve related functions. ○ Each ORF typically has its own ribosome binding site (RBS) so that ribosomes simultaneously translate ORFs on the same mRNA ○ Having multiple ORFs on a single mRNA is only possible in prokaryotes because their transcription and translation take place at the same time and in the same subcellular location. ○ The operator sequence next to the promoter is the main regulatory element in prokaryotes. Repressor proteins bound to the operator sequence physically obstructs the RNA polymerase enzyme, preventing transcription ○ Riboswitches switch between alternative secondary structures in the RNA depending on the concentration of key metabolites. The secondary structures then either block or reveal important sequence regions such as RBSs. ○ Introns are extremely rare in prokaryotes and therefore do not play a significant role in prokaryotic gene regulation. Source: WikiJournal of Medicine, 2017, 4(1), Figure Article. Available at: https://doi.org/10.15347/wjm/2017.002 MITBIO/MITADT University MITBIO/MITADT University  Prokaryotic Transcription RNA polymerase is an enzyme that transcribes DNA into RNA in prokaryotic cells. Prokaryotic cells have a single RNA polymerase that transcribes all three major classes of RNA. RNA polymerase synthesizes RNA by following a strand of DNA. Types: ○ RNA polymerase I: Transcribes rRNA ○ RNA polymerase II: Transcribes mRNA ○ RNA polymerase III: Transcribes tRNA, 5S RNA, and other small RNAs MITBIO/MITADT University  Subunits of RNA polymerase 1. α (Alpha): involved in enzyme assembly and also binds to specific DNA sequences. 2. β (Beta): forms the catalytic center, elongating the growing RNA chain. 3. β’ (Beta Prime): Together with β, it ensures accurate initiation and elongation. 4. σ (Sigma): confers precision by binding to promoter regions, marking the spot where transcription begins. MITBIO/MITADT University  Why greek letters are used in polymerase subunits? Greek letters are used to designate the subunits of RNA polymerase to provide a clear and systematic way to identify and differentiate them. Each subunit has a specific function and structure, and using Greek letters helps in categorizing these roles efficiently. MITBIO/MITADT University  Why greek letters are used in polymerase subunits? Historical Background ○ Early Discoveries The discovery of RNA polymerase and its subunits dates back to the mid-20th century. As scientists began to identify and characterize the different components of RNA polymerase, they needed a systematic way to name these subunits. ○ Greek Alphabet A simple and clear way to differentiate between multiple subunits within the same enzyme complex. This method was already in use in other areas of science, making it a familiar and convenient choice. MITBIO/MITADT University  Why greek letters are used in polymerase subunits? Historical Background ○ Early Discoveries The discovery of RNA polymerase and its subunits dates back to the mid-20th century. As scientists began to identify and characterize the different components of RNA polymerase, they needed a systematic way to name these subunits. ○ Greek Alphabet A simple and clear way to differentiate between multiple subunits within the same enzyme complex. This method was already in use in other areas of science, making it a familiar and convenient choice. MITBIO/MITADT University MITBIO/MITADT University  The DNA sequence (grey), The −35 and −10 (Pribnow box) elements are shaded yellow, the extended −10 & discriminator elements (purple) Subunits (αI, αII, ω, grey; β, light cyan; β′, light pink; Δ1.1σA, light orange) Active site Mg2+ (yellow sphere) and the nucleic acids held inside the RNAP active site channel. Brian Bae, Andrey Feklistov, Agnieszka Lass-Napiorkowska, Robert Landick, Seth A Darst (2015) Structure of a bacterial RNA polymerase holoenzyme open promoter complex eLife4:e08504. https://doi.org/10.7554/eLife.08504 MITBIO/MITADT University  Prokaryotic Transcription Bacteria have the simplest form of RNA polymerase made up of several proteins that work together to make RNA using DNA as a template RNA polymerase and the sigma factor interact the resulting group of proteins is known as the RNA polymerase ‘holoenzyme’ Transcription initiation is a major control point of gene expression. A conserved ∼400 kD catalytic core of the RNA polymerase (RNAP or E, subunit composition α2ββ′ω) ○ combines with the promoter-specificity factor σA to form the holoenzyme (EσA), ○ which locates promoter DNA and unwinds 12–14 base pairs (bps) of the DNA duplex ○ to yield the transcription-competent open promoter complex (RPo) In the presence of nucleotide substrates, RNA synthesis begins with the formation of an initial transcription complex (RPITC), transitioning to a stable elongation complex Eventually, the transcript reaches a length of around 17 nt, where σ dissociation and the transition to the stable elongation complex begins MITBIO/MITADT University MITBIO/MITADT University MITBIO/MITADT University MITBIO/MITADT University  Transcription elongation The elongation cycle comprises three basic steps: 1. Binding of a template-complementary nucleoside triphosphate (NTP) into the active site; 2. Chemical reaction of the RNA chain 3′-OH with the NTP α-PO4, catalyzed by a pair of bound Mg2+ ions, resulting in one NMP addition to the RNA and liberation of pyrophosphate; and 3. Translocation of the nucleic acid assemblage to place the next template base in the active center  Transcription elongation The elongation complex of RNAP (Figure 1) is stabilized by several sets of interactions such as: 1. downstream duplex DNA is bound within the enzyme; 2. about nine nucleotides of RNA at the growing end are annealed to the template DNA strand, forming a 9-bp RNA/DNA hybrid enclosed by protein; and 3. an additional ~5 nucleotides of RNA upstream of the hybrid are bound in a protein channel until the RNA emerges 14 nucleotides from the growing end  Transcription elongation Transcription elongation in prokaryotes is the phase of mRNA synthesis that occurs after the σ subunit dissociates from the polymerase, allowing the core enzyme to move along the DNA template. The core enzyme then synthesizes mRNA in the 5' to 3' direction Rate = 40 nt per sec. As it moves, the polymerase unwinds the DNA template ahead of it and rewinds it behind it, keeping an open region of about 17 base pairs for transcription MITBIO/MITADT University  Transcription elongation 1. DNA is continuously unwound ahead of the core enzyme and rewound behind it. 2. Base pairing between DNA and RNA is not stable hence , RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted. 3. Elongation is NOT uniform and inevitable, but modulated by regulatory influences to fast/slow/stop a. anti terminators, b. operon-specific genetic regulatory elements c. RNA can be a regulatory element. Thus, in regulation by an attenuator RNA or riboswitches d. Blocked by accident: a non coding lesion (a thymine dimer) is in the DNA template or clashes in replication and recombination. Here transcription complex is removed. Roberts, J. W., Shankar, S., & Filter, J. J. (2008). RNA polymerase elongation factors. Annual review of microbiology, 62, 211–233. https://doi.org/10.1146/annurev.micro.61.080706.093422 MITBIO/MITADT University  Transcription elongation Polymerase is accurate (?) - only about 1 error in 10,000 bases (error rate is OK, since many transcripts are made from each gene) Elongation rate is 20-50 bases per second - slower in G/C-rich regions (why??) and faster elsewhere Topoisomerases precede and follow polymerase to relieve supercoiling  Termination of prokaryotic transcription Transcription can terminate in two ways: rho-dependent or rho-independent. ○ Rho-dependent termination occurs when the rho protein collides with the polymerase at a stretch of G nucleotides near the end of the gene. ○ Rho-independent termination occurs when the polymerase stalls at a stable hairpin formed by complementary C–G nucleotides at the end of the mRNA Roberts, J. W., Shankar, S., & Filter, J. J. (2008). RNA polymerase elongation factors. Annual review of microbiology, 62, 211–233. https://doi.org/10.1146/annurev.micro.61.080706.093422 MITBIO/MITADT University MITBIO/MITADT University MITBIO/MITADT University MITBIO/MITADT University Eukaryotic transcription Prokaryotes and eukaryotes both perform transcription similarly, but with key differences. Eukaryotic mRNA synthesis is much more complex than in prokaryotes. Eukaryotes have a membrane-bound nucleus and organelles. Eukaryotic cells must transport mRNA to the cytoplasm and protect it from degradation before translation. Eukaryotes have three polymerases, each with over 10 subunits, compared to the single five-subunit polymerase in prokaryotes. Additionally, each eukaryotic polymerase needs a unique set of transcription factors to bind to the DNA template. Their mRNAs are typically monogenic, specifying a single protein.  Eukaryotic transcription RNA polymerase I is found in the nucleolus, where it transcribes rRNA, except for 5S rRNA. These rRNAs are structural and essential for translation. RNA polymerase II, located in the nucleus, synthesizes protein-coding pre-mRNAs, which undergo extensive processing before translation. RNA polymerase III, also in the nucleus, transcribes 5S pre-rRNA, pre-tRNAs, and small nuclear pre-RNAs. These small RNAs play roles in translation and splicing. The sensitivity of these polymerases to α-amanitin, a toxin from the Death Cap mushroom, varies: RNA polymerase I is insensitive, RNA polymerase II is highly sensitive, and RNA polymerase III is moderately sensitive. This sensitivity helps researchers identify which polymerase transcribes a gene. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  Eukaryotic RNA Polymerases and General Transcription Factors Cellular Product of α-Amanitin RNA Pol Compartment Transcription Sensitivity All rRNAs except 5S I Nucleolus Insensitive rRNA All protein-coding Extremely II Nucleus nuclear pre-mRNAs sensitive 5S rRNA, tRNAs, and small Moderately III Nucleus nuclear RNAs sensitive Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf Type of RNA synthesized RNA polymerase Nuclear genes mRNA II tRNA III ~Some small nuclear (sn) and small rRNA cytoplasmic (sc) RNAs are transcribed by polymerase II and others by polymerase III. 5.8S, 18S, 28S I 5S III *The mitochondrial and chloroplast RNA polymerases are similar to bacterial snRNA and scRNA II and III~ enzymes. Mitochondrial genes Mitochondria Chloroplast genes Chloroplast* https://www.ncbi.nlm.nih.gov/books/NBK9935/  Eukaryotic transcription RNA polymerase I is found in the nucleolus, where it transcribes rRNA, except for 5S rRNA. These rRNAs are structural and essential for translation. RNA polymerase II, located in the nucleus, synthesizes protein-coding pre-mRNAs, which undergo extensive processing before translation. RNA polymerase III, also in the nucleus, transcribes 5S pre-rRNA, pre-tRNAs, and small nuclear pre-RNAs. These small RNAs play roles in translation and splicing. The sensitivity of these polymerases to α-amanitin, a toxin from the Death Cap mushroom, varies: RNA polymerase I is insensitive, RNA polymerase II is highly sensitive, and RNA polymerase III is moderately sensitive. This sensitivity helps researchers identify which polymerase transcribes a gene. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  Structure of an RNA Polymerase II Promoter Eukaryotic promoters are larger and more complex than prokaryotic ones, but both contain a TATA box. In the mouse thymidine kinase gene, the TATA box is around -30 from the initiation site and has the sequence TATAAAA. This sequence, while different from the E. coli TATA box, maintains the A-T rich element, aiding in DNA unwinding for transcription. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  A generalized promoter of a gene transcribed by RNA polymerase II. Transcription factors recognize the promoter. RNA pol II then binds and forms the transcription initiation complex. MITBIO/MITADT University  Transcription Factors for RNA Polymerase II Eukaryotic transcription involves more than just polymerases and promoters. Basal transcription factors, enhancers, and silencers regulate pre-mRNA synthesis. Enhancers and silencers boost transcription efficiency but aren't essential. Basal transcription factors, named TFII (A–J), form a preinitiation complex that recruits RNA polymerase II. RNA polymerases I and III use fewer transcription factors, but the process is similar. Eukaryotic transcription is tightly regulated, requiring various proteins to interact with DNA. This complex process ensures precise pre-mRNA synthesis for protein production. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  Transcription Factors for RNA Polymerase II Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  Transcription Factors for RNA Polymerase II Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  Transcription Factors for RNA Polymerase II Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  The Evolution of Promoters Gene evolution through mutations during DNA replication can alter functions or features, potentially benefiting the cell. Similarly, eukaryotic promoters and regulatory sequences can evolve. If a gene becomes more valuable, its promoter may evolve to recruit transcription factors more efficiently, boosting gene expression. Research on promoter evolution shows mixed results due to the difficulty in defining promoter boundaries. Some promoters are within genes, others far upstream or downstream. However, studies on human core promoters show they evolve faster than protein-coding genes. The impact of promoter evolution on higher organisms remains unclear, but it offers an intriguing alternative to gene evolution. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  Promoter Structures for RNA Pol I and III In eukaryotes, the conserved promoter elements differ for genes transcribed by RNA polymerases I, II, and III. RNA polymerase I transcribes genes that have two GC-rich promoter sequences in the -45 to +20 region. Another promoter with additional sequences in the region from -180 to -105 upstream of the initiation site will further enhance initiation. Genes that are transcribed by RNA polymerase III have upstream promoters or promoters that occur within the genes themselves. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  Eukaryotic Transcription initiation During initiation, RNA polymerase recognizes a promoter site upstream of the gene and unwinds the DNA. Most RNA polymerase II promoters have a TATA box around -25 from the start site, aiding in positioning the enzyme for transcription. The TATA box sequence is similar to the -10 sequence in prokaryotes and helps in DNA unwinding. Some genes lack a TATA box and use an initiator element instead. RNA polymerase II requires general transcription factors (TFII) to form a preinitiation complex. TFIID binds to the TATA box, followed by TFIIA, TFIIB, RNA polymerase II (with TFIIF), TFIIE, and TFIIH, forming the transcription initiation complex. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://microbenotes.com/eukaryotic-transcription/  Eukaryotic Transcription initiation During initiation, RNA polymerase recognizes a promoter site upstream of the gene and unwinds the DNA. Most RNA polymerase II promoters have a TATA box around -25 from the start site, aiding in positioning the enzyme for transcription. The TATA box sequence is similar to the -10 sequence in prokaryotes and helps in DNA unwinding. Some genes lack a TATA box and use an initiator element instead. RNA polymerase II requires general transcription factors (TFII) to form a preinitiation complex. TFIID binds to the TATA box, followed by TFIIA, TFIIB, RNA polymerase II (with TFIIF), TFIIE, and TFIIH, forming the transcription initiation complex. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://microbenotes.com/eukaryotic-transcription/ Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://microbenotes.com/eukaryotic-transcription/  Eukaryotic Elongation TFIIH has two main functions: 1. It acts as a helicase, using ATP to unwind the DNA helix for transcription to begin. 2. It phosphorylates RNA polymerase II, causing it to change shape and dissociate from the initiation complex. Phosphorylation occurs on the C-terminal domain (CTD) of RNA polymerase II. Only non-phosphorylated RNA polymerase II can initiate transcription, while only phosphorylated RNA polymerase II can elongate RNA. RNA polymerase II then moves along the DNA, synthesizing RNA in the 5' to 3' direction. The RNA produced from a protein-coding gene is called a primary transcript. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  Eukaryotic Termination Elongation continues until termination. Unlike prokaryotic RNA polymerase, RNA polymerase II doesn't stop at a specific site but can terminate at varying distances downstream of the gene. It lacks specific termination signals and can transcribe RNA from a few to thousands of base pairs past the gene's end. The transcript is cleaved internally before RNA polymerase II finishes, releasing the upstream portion as the initial RNA (pre-mRNA for protein-coding genes). The remaining transcript is digested by a 5′-exonuclease (Xrn2 in humans). When the exonuclease catches up to RNA polymerase II, it helps disengage the polymerase, terminating transcription. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  RNA processing The primary eukaryotic mRNA transcript, also known as hnRNA or pre-mRNA, is longer and located in the nucleus. It undergoes steps to become mature RNA: 1. Cleavage: Larger RNA precursors are cleaved into smaller RNAs. The primary transcript is cleaved by ribonuclease-P to form 5-7 tRNA precursors. 2. Capping and Tailing: A cap (7-methyl guanosine) is added to the 5′ end, and a poly-A tail is added to the 3′ end. The cap is a modified guanosine triphosphate (GTP) molecule. Eukaryotic primary mRNAs consist of non-coding introns and coding exons. Introns are removed through RNA splicing, which uses ATP to cut the RNA, releasing introns and joining exons to form mature mRNA. 1. Nucleotide Modifications: Common in tRNA, including methylation (e.g., methyl cytosine), deamination (e.g., inosine from adenine), dihydrouracil, and pseudouracil. Post-transcription processing is essential to convert the primary transcript into functional RNAs. Sources: 1. Cooper GM. The Cell: A Molecular Approach. 2nd edition; 2000. Eukaryotic RNA Polymerases and General Transcription Factors. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9935/ 2. https://bio.libretexts.org/@go/page/1898?pdf  Prokaryotic and Eukaryotic transcription MITBIO/MITADT University  Prokaryotic and Eukaryotic transcription MITBIO/MITADT University  Prokaryotic and Eukaryotic transcription MITBIO/MITADT University  Prokaryotic and Eukaryotic transcription MITBIO/MITADT University  Prokaryotic and Eukaryotic transcription MITBIO/MITADT University  Prokaryotic and Eukaryotic transcription MITBIO/MITADT University  Prokaryotic and Eukaryotic transcription MITBIO/MITADT University  Prokaryotic and Eukaryotic transcription MITBIO/MITADT University Post transcriptional Modifications  5’ capping 7-methylguanosine (a modified nucleotide at the 5′ end) residues connected by a 5′-5′ triphosphate bridge, is regulated and essential for the production of stable and mature messenger RNA Functions: Protection: from degradation by exonucleases. Translation Initiation: recognized by the ribosome, translation initiation factors Absent in “G” Transport: aids in the export of mRNA from nucleus to cytoplasm. Capping Process: A. RNA Triphosphatase: Removes one phosphate from the 5’ end of the nascent RNA. B. Guanylyltransferase: Adds a guanine monophosphate (GMP) to the 5’ end, creating a 5’ to 5’ triphosphate bridge. C. Methyltransferase: Methylates the guanine at the 7th position. Types of Caps: A. Cap-0: The basic structure with a 7-methylguanosine. B. Cap-1: Additional methylation at the 2’ position of the first nucleotide. C. Cap-2: Further methylation at the 2’ position of the second nucleotide. 7-methylguanosine  5’ capping MITBIO/MITADT University  Poly-A Tailing Addition of a multiple adenosine monophosphates, forming a stretch of RNA (poly(A) tail) to the 3’ end, typically of messenger RNA (mRNA) molecule Functions: 1. Stability: protects mRNA from degradation by exonucleases. 2. Translation: increases translation efficiency by aiding in the recruitment of ribosomes. 3. Nuclear Export: export of mRNA from the nucleus to the cytoplasm. Polyadenylation Process: 1. Cleavage: The 3’ end of the pre-mRNA is cleaved by CF-I & CF-II (Pre-mRNA cleavage factor) 2. Addition: Poly(A) polymerase adds adenine nucleotides to the cleaved 3’ end. Regulation: 1. Alternative Polyadenylation: Different polyadenylation sites can be used within the same gene, leading to the production of mRNA variants with different 3’ untranslated regions (UTRs) 2. Cell Type Specificity: The choice of polyadenylation sites can vary between different cell types and conditions MITBIO/MITADT University MITBIO/MITADT University  Polyadenylation steps: 1. The processive polyadenylation complex binds to precursor mRNA 2. CPSF binds the complex 10–30 nucleotides downstream and cleaves the 3′-most portion 3. Polyadenylate polymerase enzyme polyadenylates the resulting end to form the poly(A) tail by cleaving pyrophosphate from ATP & adding AMP units to the RNA. 4. When the poly(A) tail reaches around 250 nucleotides in length, the enzyme loses its ability to bind to CPSF and polyadenylation ceases, hence dictating the length of the poly(A) tail. 5. CPSF interacts with RNA polymerase II, to stop transcription. 6. RNA polymerase II encounters a “termination sequence” (5’TTTATT3′ on the DNA template and 5’AAUAAA3′ on the primary transcript).  RNA Splicing A process of mature mRNA formation by pre-mRNA processing by removing introns and joining exons 1. Spliceosome Complex: a. a large and complex molecular machine, is responsible for the splicing process. It consists of small nuclear RNAs (snRNAs) and a variety of associated protein factors. b. Key snRNAs involved include U1, U2, U4, U5, and U6, each playing a role in different stages of splicing. 2. Splicing Sites: a. Splicing occurs at specific sequences called splice sites. The 5' splice site marks the beginning of an intron, while the 3' splice site marks the end. b. The branch point, located within the intron, is crucial for the formation of the lariat structure during splicing. MITBIO/MITADT University MITBIO/MITADT University MITBIO/MITADT University MITBIO/MITADT University  RNA Splicing 3. Splicing Mechanism: a. The process begins with the recognition of the 5' splice site by the spliceosome. b. The 5' end of the intron is cut, and the lariat structure is formed by the attachment of the 5' end to the branch point. c. The 3' end of the intron is then cut, and the exons are joined together, releasing the intron as a lariat. 4. Alternative Splicing: a. Alternative splicing allows a single gene to produce multiple mRNA variants by including or excluding different exons. b. This increases the diversity of proteins that can be produced from a single gene and plays a key role in regulating gene expression and development.  RNA Splicing 3. Regulation of Splicing: a. Splicing is tightly regulated by various splicing factors and regulatory elements that influence which splice sites are used. b. Regulatory proteins can enhance or repress splicing of specific exons or introns, contributing to the regulation of gene expression and cellular responses. 4. Disease Associations: a. Aberrations in splicing can lead to various diseases, including some types of cancer and genetic disorders such as cystic fibrosis and Duchenne muscular dystrophy. b. Mutations in splice sites or splicing factors can disrupt normal splicing, leading to the production of faulty or non-functional proteins. References: Fine Structure of Prokaryotic and Eukaryotic Genes: Shafee, T., & Lowe, R. (2016). Eukaryotic and Prokaryotic Gene Structure. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3013506 Structure and Function of Promoters in mRNA, rRNA, tRNA Genes: Shine, M., Gordon, J., Schärfen, L., Zigackova, D., Herzel, L., & Neugebauer, K. M. (2024). Co-transcriptional gene regulation in eukaryotes and prokaryotes. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3013506 RNA Polymerases in Prokaryotes and Eukaryotes: Types and Function: RNA Transcription by RNA Polymerase: Prokaryotes vs Eukaryotes.https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3013506. Transcription of mRNA, rRNA, and tRNA Genes in Prokaryotes and Eukaryotes: Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular Biology of the Cell (4th ed.). Garland Science. Chapter 6: “Transcription of RNA”. Post-Transcriptional Processing of mRNA – 5’ Capping, Splicing, Polyadenylation, and RNA Editing: Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., Bretscher, A., Ploegh, H., Amon, A., & Martin, K. C. (2016). Molecular Cell Biology (8th ed.). W.H. Freeman. Chapter 10: “RNA Processing”. MITBIO/MITADT University Interesting Links: 1. Fine Structure of Prokaryotic and Eukaryotic Genes: ○ https://www.youtube.com/watch?v=xTnNv7YplSohttps://www.bing.com/videos/search?view=detail&q=cture+and+ Function+of+Promoters+in+mRNA%2c+rRNA%2c+tRNA+Genes+videos&&mid=11C3BFC986DFAE5743C711C3 BFC986DFAE5743C7&&FORM=VRDGAR 2. Structure and Function of Promoters in mRNA, rRNA, tRNA Genes: ○ https://www.bing.com/videos/search?view=detail&q=cture+and+Function+of+Promoters+in+mRNA%2c+rRNA%2c +tRNA+Genes+videos&&mid=EF2ECA327348B98CC336EF2ECA327348B98CC336&&FORM=VRDGAR 3. RNA Polymerases in Prokaryotes and Eukaryotes: Types and Function: ○ https://www.bing.com/videos/search?view=detail&q=NA+Polymerases&&mid=05D1AAA02552B61A50DF05D1AA A02552B61A50DF&&FORM=VRDGAR 4. Transcription of mRNA, rRNA, and tRNA Genes in Prokaryotes and Eukaryotes: ○ https://www.bing.com/videos/search?view=detail&q=NA+Polymerases&&mid=A55462E3F7957AAA1D09A55462E 3F7957AAA1D09&&FORM=VRDGAR ○ https://www.bing.com/videos/search?view=detail&q=NA+Polymerases&&mid=74808E17922E5F01E88574808E17 922E5F01E885&&FORM=VRDGAR 5. Post-Transcriptional Processing of mRNA – 5’ Capping, Splicing, Polyadenylation, and RNA Editing: ○ https://www.bing.com/videos/search?view=detail&q=Post-Transcriptional+Processing+of+mRNA+&&mid=D3D1EE 947EFF052C06D5D3D1EE947EFF052C06D5&&FORM=VRDGAR ○ https://www.bing.com/videos/search?view=detail&q=Post-Transcriptional+Processing+of+mRNA+&&mid=E0A191 A87B131368A2E0E0A191A87B131368A2E0&&FORM=VRDGAR MITBIO/MITADT University Additional Information: Touchon, M., & Rocha, E. P. (2016). Coevolution of the Organization and Structure of Prokaryotic Genomes. Cold Spring Harbor perspectives in biology, 8(1), a018168. https://doi.org/10.1101/cshperspect.a018168 Brown TA. Genomes. 2nd edition. Oxford: Wiley-Liss; 2002. Chapter 2, Genome Anatomies. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21120/ Berg, M. D., & Brandl, C. J. (2021). Transfer RNAs: diversity in form and function. RNA biology, 18(3), 316–339. https://doi.org/10.1080/15476286.2020.1809197 Pan, T. Modifications and functional genomics of human transfer RNA. Cell Res 28, 395–404 (2018). https://doi.org/10.1038/s41422-018-0013-y Corbett A. H. (2018). Post-transcriptional regulation of gene expression and human disease. Current opinion in cell biology, 52, 96–104. https://doi.org/10.1016/j.ceb.2018.02.011 Zhao, B., Roundtree, I. & He, C. Post-transcriptional gene regulation by mRNA modifications. Nat Rev Mol Cell Biol 18, 31–42 (2017). https://doi.org/10.1038/nrm.2016.132 Giudice, J., Jiang, H. Splicing regulation through biomolecular condensates and membraneless organelles. Nat Rev Mol Cell Biol 25, 683–700 (2024). https://doi.org/10.1038/s41580-024-00739-7 MITBIO/MITADT University Question Bank: Fine Structure of Prokaryotic and Eukaryotic Genes 1. What are differences between the fine structure of prokaryotic and eukaryotic genes? 2. How do introns and exons contribute to the complexity of eukaryotic genes? 3. Describe the role of operons in prokaryotic gene regulation. 4. What is the significance of the TATA box in eukaryotic gene promoters? Structure and Function of Promoters in mRNA, rRNA, and tRNA Genes 1. How do promoters differ between mRNA, rRNA, and tRNA genes in prokaryotes? 2. What are the core promoter elements in eukaryotic mRNA genes? 3. Explain the role of upstream promoter elements in the transcription of rRNA genes. 4. How do enhancers and silencers affect promoter activity in eukaryotic genes? MITBIO/MITADT University Question Bank: RNA Polymerases in Prokaryotes and Eukaryotes: Types and Functions 1. Compare the types and functions of RNA polymerases in prokaryotes and eukaryotes. 2. What is the role of RNA polymerase I in eukaryotic cells? 3. How does RNA pol II differ from RNA polymerase III in terms of their transcriptional targets? 4. Describe the mechanism of transcription initiation by RNA polymerase in prokaryotes. Transcription of mRNA, rRNA, and tRNA Genes 1. Outline the steps involved in the transcription of mRNA in eukaryotic cells. 2. How is the transcription of rRNA genes regulated in prokaryotes? 3. What are differences in the transcription of tRNA genes in prokaryotes and eukaryotes? 4. Explain the role of transcription factors in the transcription of eukaryotic mRNA genes. MITBIO/MITADT University Question Bank: Post-Transcriptional Processing of mRNA 1. What is the significance of 5’ capping in mRNA processing? 2. Describe the mechanism of splicing and its importance in mRNA maturation. 3. Explain the process of polyadenylation and its role in mRNA stability. Application and Analysis 1. Analyze the impact of a mutation in the promoter region of a prokaryotic gene on its expression. 2. Compare the efficiency of transcription between prokaryotic and eukaryotic systems and discuss the factors influencing it. 3. Evaluate the consequences of defective splicing on gene expression and protein function. MITBIO/MITADT University MIT School of Bioengineering Sciences and Research (A Constituent unit of MIT ADT University) Rajbaug Campus, Next to Hadapsar, Loni Kalbhor, Maharashtra 412201