MBG Block 3 Review Slides PDF

Exam 3 Review BMS MOLECULAR BIOLOGY AND GENETICS NOTE: This is a basic review guide and does not contain every single detail that BLOCK 3: MOLECULAR GENETICS might be required for the examination. It...

Exam 3 Review BMS MOLECULAR BIOLOGY AND GENETICS NOTE: This is a basic review guide and does not contain every single detail that BLOCK 3: MOLECULAR GENETICS might be required for the examination. It is meant to serve as a quick guide to assist your studies especially in the final days of studying. Some Terminology and Key Phrases Molecular Genetics, Central Dogma of Molecular Deamination, abasic/ap sites, oxidative damage, Genetics ionizing radiation, UV radiation, polycyclic aromatic hydrocarbons (PAH), aromatic amines, alkylating Genome, Exome, Transcriptome, Proteome, agents Metabolome Interstrand crosslinks, intrastrand crosslinks, Variation, Transmissibility/transmission mismatch Replication, Transcription, Translation Higher order structures Nucleases, aminoacyl synthetases, peptidyl Structural vs regulatory RNAs; coding vs noncoding transferases, methyltransferases RNAs Origin Licensing, Origin Firing, Pre-RC, Pre-IC Polycistronic vs monocistronic; Colinear vs split Mutation: Silent, missense, nonsense, RNA Editing, Capping, polyadenylation, splicing, transition, transversion spliceosome, snRNA, snRNP Objectives: Introduction 1. Define the following terms using both technical and working definitions: Molecular Genetics, Genome, Transcriptome, Proteome, Polypeptide, Metabolome, Genotype, and Phenotype 2. Summarize the central dogma of molecular genetics and explain the conceptual hierarchy of molecular genetics in terms of how and when genetic information is observed phenotypically 3. Explain the relationship between DNA, RNA, and protein in terms of how each influences and interacts with one another 4. Explain how molecular analyses contribute to our understanding of biological function with emphasis on “omics” and their role in diagnostics  Objectives: Biochemistry of DNA 5. List the criteria for genetic information and explain why genetic material must meet this criteria 6. Describe the structure of DNA including base-pairing, directionality, and complementarity based on the Watson-Crick Model 7. Explain the forces involved in generating the stability of the double-stranded DNA molecule and how this compares to single-stranded alternatives 8. Summarize the 3 forms of DNA discussed (B, A, and Z) and explain the implications of each form on function 9. Explain how DNA sequences correlate with DNA structure/form and stability of the overall molecule 10. Compare and Contrast double-stranded and single-stranded DNA in terms of their stability and replication 11. Define the term C-value and explain the C-value paradox 12. Assess the consequences for changes (typical variation and mutation) in DNA sequence on DNA structure, mRNA product, protein product, and overall gene function and link these changes to human variation or disease** **This is a major aim and theme for the entire block  Objectives: DNA Replication 1. Diagram a basic bidirectional replication fork from an origin of replication and summarize the steps of Replication from Origin of Replication through FLAP Endonuclease activity with particular emphasis on the molecules involved: Primase, RNA Primer, DNA polymerase (all forms discussed), Helicase, Ligase, Topoisomerase 2. Explain the process of origin firing and the role of multiple origins of replication in human replication and compare and contrast the activities and features of early and late firing origins of replication 3. Explain the process of origin licensing and firing and assess the consequences for alterations in the activity for the origin recognition complex, components of the pre-replication complex, and components of the pre-initiation complex (ORC, Pre-RC, Pre-IC) 4. Compare and contrast leading and lagging strand synthesis with emphasis on the molecules involved in each and the movement of the polymerase vs base addition 5. Outline the biochemical process that enables the addition of new nucleotides with emphasis on required biochemical functional groups and strand directionality and assess the consequence for lack of or loss of the 3’ hydroxyl group 6. Compare and contrast replication in circular dsDNA, in ssDNA (circular or linear), and linear dsDNA and Explain the cause of the limitation in the replication of linear chromosomes and the corresponding consequence (end-replication problem) 7. Compare and contrast types of polymerases in terms of their roles in the processes of replication and assess the consequence for errors in their activity 8. Compare and contrast topoisomerase I and topoisomerase II and assess the consequence for errors in their activity 9. Assess the consequences for replication and DNA sequence for errors in replication and in the activity of the replication machinery (corresponds to all slides) Replication: A quick summary Key Molecules of Replication ◦ Primase ◦ Topoisomerase I and II ◦ Helicase ◦ Single strand binding proteins ◦ Polymerases ◦ DNA ligase ◦ Flap endonuclease and DNA2 nuclease Additional Features ◦ Origin of Replication ◦ Pre-replication complex (ORC, cdc6, cdt1, MCM helicase) and Pre-Initiation complex (formation of replication fork and association of replication machinery) ◦ Direction of Synthesis vs Direction of Polymerase movement ◦ Leading vs Lagging strands ◦ Okazaki Fragments  The Process of Initiation of Replication The 2 Stages of Replication Initiation (perspectives from yeast) 1. Origin Licensing 2. Origin Firing (step immediately following formation of Pre-IC) Licensing can be thought of as a 2 step process: ◦ Origin (ORI) recognition and formation of the Pre-RC ◦ Identification of the time to fire and formation of the Pre-IC Firing Analogy: ◦ Pre-RC = loading an arrow ◦ Pre-IC = pulling back on the bow string ◦ The arrow can’t fly without both and loading an arrow (form the Pre-RC) doesn’t guarantee that you will prep to fire (form the Pre-IC) ◦ The arrow leaving can be viewed as actually firing which cannot happen until you pull the string taunt (form the Pre-IC) Gnan et al 2020 End Replication Problem Finalize Okazaki Fragments Replication (and transcription) Biochemistry and Elongation All synthesis is 5’ to 3’ while reading and moving on a template 3’ to 5’ ◦ DUE TO 3’ HYDROXYL ◦ Dideoxynucleotide variants that lack the 3’ hydroxyl will stall replication (AND transcription) ◦ Elongation will cease (can lead to replication fork collapse or bypass) In Replication of dsDNA, due to limitation in synthesis, there are two types of strand synthesis ◦ Leading (polymerase moves seamlessly in same direction as the replication fork) ◦ Lagging (polymerase movement disjointed b/c in opposite direction of replication fork movement; Okazaki Fragments)  Objectives: RNA Structure and Transcription 1. Compare and contrast structure and sequence of DNA and RNA and compare and contrast eukaryotic and prokaryotic mRNA 2. Explain how RNA forms higher order structures and identify sequences that would be capable of forming these structures 3. Summarize the types of RNA discussed and their category of corresponding functions if known 4. Explain what is meant by polycistronic mRNA and how it relates to cellular activity 5. Explain the role of promotors and their importance to gene transcription (additional regulatory elements will be addressed in a later section) 6. Summarize the process of Transcription (with an emphasis on the broad process steps of Initiation, Elongation, and Termination) ◦ Explain what is meant by sense and antisense strands (coding and template strands) 7. Explain the mechanics of Transcription initiation and termination and Compare and contrast prokaryotic and eukaryotic transcription initiation and termination ◦ Explain the overall role of transcription factors, promoters, and termination sequences in the process of Transcription 8. Identify the activity of the different RNA polymerases and how termination of their activity varies 9. Assess the consequence for changes in DNA or RNA sequence, mRNA structure, RNA polymerase activity, or transcription factor activity on the process of transcription and the final products generated* *This aim will be continued in future lectures Transcription: A quick summary Key Molecules of Transcription ◦ RNA polymerase (compare and contrast: Pol I, II, III) ◦ Transcription Factors ◦ Rho-dependent vs Rho-independent ◦ Release Factors Additional Features ◦ Promoter ◦ TATA box and Consensus sequence ◦ Termination Sequence ◦ Coding vs Template  Objectives: Gene Code and Translation 1. Explain the role of a codon in terms of the genetic code and protein structure 2. Explain the role of tRNA in the process of translation and summarize the structural elements of tRNA essential to its function 3. Summarize the process of Translation (with an emphasis on the activity of the ribosome and the broad process steps of Initiation, Elongation, and Termination) 4. Compare and contrast prokaryotic and eukaryotic translation with emphasis on ribosomes and release factors 5. Compare and contrast Replication, Transcription, and Translation* 6. Identify the sequences of either nucleotides or amino acids that would be generated from each of the following situations (will be provided a sequence of nucleotides) ◦ Following DNA Replication with and without errors/mutation ◦ In Transcription if provided either the Coding (sense) or Template (anti-sense) Strands ◦ In Translation if provided an mRNA transcript sequence ◦ From either Replication or Transcription if incorporation of an altered base occurs ◦ Identify the mRNA transcript or DNA sequence from a sequence of amino acids or primary protein structure (work backwards from amino acids back to RNA and then DNA) 7. Explain the consequences in terms of sequence changes, structure, and function for the final products (RNA or Protein) for each of the following: replication errors, strand slippage errors, errors in splicing, and induction of mutations to one or more bases that fail to undergo repair ◦ Identify the sequence changes in DNA, RNA, and protein expected in transition and transversion errors ◦ Define the consequence in terms of protein structure for mutations that fail to be repaired or undergo aberrant repair ◦ Identify the cause of a given phenotype/change in functional product* Translation: A quick summary Key Molecules of Translation ◦ Ribosome ◦ Small vs large subunit ◦ Peptidyl transferase ◦ tRNA ◦ Aminoacyl synthetases ◦ rRNA ◦ Release Factors Additional Features ◦ Codon vs anticodon ◦ Shine-Delgano Sequence ◦ EPA Gene Code  Objectives: DNA Mutation and Repair 1. Explain what is meant by the term mutation and summarize how mutations can be classified (identify types of mutation and compare/contrast them) 2. Describe the consequences of mutations with particular emphasis on additional mutations and how they can be either more deleterious or beneficial/complementary 3. Identify the structural similarities and differences in bases with emphasis on the common and rare forms (keto vs enol & imino vs amino) and explain the role of tautomeric shift and spontaneous deamination in the generation of replication error 4. Explain the consequences for errors during replication in terms of DNA structure, future replication, DNA nucleotide sequences, and eventual protein products 5. Compare and contrast silent, missense, nonsense, and frameshift mutations in terms of changes in the DNA sequence and the corresponding protein products** 6. Compare and contrast mechanisms of endogenous DNA damage and the types of mutation induced 7. Compare and contrast mechanisms of exogenous DNA damage and the types of mutation induced by the agent including: ionizing radiation, UV damage, alkylating agents, and polycyclic aromatic hydrocarbons. 8. List the repair mechanisms and what types of damage they repair 9. Summarize the steps involved in the following repair mechanisms and explain the potential consequences of repair: Base-excision repair, nucleotide excision repair, mismatch repair, interstrand crosslink repair, homologous recombination, and nonhomologous end-joining ◦ The emphasis should NOT be on knowing each and every component of each process but rather on key molecules involved in the following key steps: recognition of damage, removal of damage, replacement of removed bases, and sealing of DNA backbone **This aim will be revisited in each process lecture DNA Damage Common vs Rare forms of nucleotides and pairing arrangements ◦ Keto v. Enol Amino v. Imino Endogenous vs Exogenous damage Damage Consequences ◦ Frame-shift mutation, Silent, Missense, Nonsense ◦ Breaks, intrastrand crosslinks, interstrand crosslinks, abasic sites, etc…  Repair What is Recognition Factors or Removal of Replacement of Conclusion Mechanism repaired? Key Feature Damage bases Direct Base alterations Photolyase Not removed; base N/A Repaired base DNA Alkyltransferase chemically repaired rehybridizes; backbone never Repair cut BER Damaged DNA N Glycosylases remove Glycosylase removes Repair Polymerase Ligase seals : base/nucleotide base; damage (single base); backbone WITHOUT helical Requires AP Endonuclease Endonuclease cleaves distortion (APE) displaced base FYI Bulky, helical Uvr molecules (prok); XP backbone Can be global or Repair Polymerase Ligase seals NER Detailed distorting lesions proteins (eukary) transcription coupled; multiple backbone Summar bases removed y MMR Base mismatches MSH (prok) and MLH/PMS (eukary) Nuclease removal of bases (multiple) Repair polymerase Ligase seals backbone ICL Interstrand FANC proteins; BRCA1 Nuclease removal of Repair Polymerase Ligase seals crosslinks (stalled bases (multiple) with HR backbone rep. forks) HR Single and double FANC proteins, BRCA1, and Not removed; end Repair Polymerase Ligase seals strand breaks RAD51; homology search processing using homologous backbone and strand invasion template NHEJ Double strand Ku70/80 heterodimers; Not removed; end Repair polymerase Ligase seals breaks End processing via processing backbone exonucleases  Repair What is repaired? Recognition Factors or Key Mechanism Feature Direct Base alterations Repairs Base Only (without cleavage); chemical repair DNA Repair BER Damaged base/nucleotide WITHOUT helical distortion DNA N Glycosylases remove base; Requires AP Endonuclease (APE) :Basic Starts base first then sugar removal summary NER Bulky, helical distorting lesions Can be coupled to transcription and including some crosslinks other repair mechanisms with most of MMR Base mismatches Requires recognition of mismatch to the occur and can fix the wrong base expected ICL Interstrand crosslinks (stalled rep. forks) May be coupled to NER to remove the DNA adduct formed by repair details HR Single and double strand breaks homology search and strand invasion NHEJ Double strand breaks Ku70/80 heterodimers; End processing via exonucleases  Objectives: RNA Processing and Splicing 1. Define the following terms or processes: deamination, colinear vs split, polyadenylation, methyltransferase, exon, intron, lariat, snRNA, snRNP, spliceosome, upstream elements, downstream elements, and regulatory elements 2. Identify the consequence of base deamination and Explain the role of RNA modification/RNA Editing in generating unique protein products 3. Compare and contrast RNA processing and editing in prokaryotes vs. eukaryotes 4. Describe the basic structure of RNA molecules with emphasis on processed Eukaryotic mRNA and the components added to generate the final version exported from the nucleus 5. Explain the role of mRNA processing in eukaryotes and list the stages and factors involved in mRNA processing ◦ Explain the importance of mRNA capping and the role of methyltransferases in mRNA capping 6. Compare and contrast snRNA and mRNA in terms of gene structure, elements located within the DNA, and processing 7. Explain the role of snRNPs in the spliceosome and identify the role in splicing for each of the following: U1, U2, U4, U5, and U6 8. Summarize the stages of major spliceosome assembly and splicing in eukaryotes with emphasis on the activity of the snRNPs, helicases (Prp5, Sub2, and Prp22), Complexes (A, B, and C), and post-spliceosomal complex 9. Summarize and explain alternative splicing, its regulation, and its role in increasing functional diversity in biological systems  mRNA Capping via Methyltransferases Forms on the first nucleotides of RNA transcribed by RNA pol II Variable structure across organisms ◦ In mammals main form = m7G(5ʹ)ppp(5ʹ)Xm ◦ 7-methylguanosine (m7G) is linked to the first transcribed nucleotide (X) via a 5ʹ to 5ʹ triphosphate bridge CAPAM is a capping methyltransferase that specifically caps adenosine nucleotides in the first position (Xm) Protect mRNA from nucleases and innate immune responses LO5 Cowling 2019  Compare and Contrast snRNA and mRNA snRNA vs mRNA ◦ Both are processed ◦ Both contain elements required for transcription that are similar but NOT identical ◦ The sequences of DNA create different structural elements that enable the same activity (transcription start and stop)  Objectives: Introduction to Gene Regulation 1. Define the following terms or phrases: constitutive, inducer or activator, repressor or silencer, inhibitor, co-repressor, negative regulation, positive regulation, regulatory element, transposition, transposable element, and insertional mutagenesis 2. Explain the role of gene regulation in cellular and organismal function 3. Compare and contrast positive and negative gene regulation and Explain the role of small effector molecules in gene regulation with emphasis on the differences between induction and repression of gene activity 4. Compare and contrast gene regulation by prokaryotes and eukaryotes 5. Summarize the regulatory elements of eukaryotic genes and their role in regulation of transcription with emphasis on initiation of transcription and the role of 3D space in transcription initiation 6. Explain the role of transcription factors and regulatory elements in gene regulation in terms of structural rearrangements or other changes in availability of key regulatory elements 7. Summarize the process of DNA transposition and assess the consequences for aberrant transposition such as viral- mediated DNA transposition 8. Summarize the process of V(D)J recombination with emphasis on the RAG complex and explain the connections with transposition 9. Assess the consequences for gene activity for changes in sequences of or access to regulatory elements and their associated proteins  Prokaryotic Gene Regulation: Small Effector Molecules Additional small effector molecules play a role in gene regulation ◦ Do not bind DNA directly but rather bind the regulatory proteins ◦ Induces conformational change in regulatory protein altering its binding to DNA ◦ Requires sufficient concentration to carry out effect: Concentration Dependent ◦ Defined by how they effect transcription/Classified by activity ◦ Can function allosterically ◦ Regulate by binding to sites other than active site ◦ Bind to allosteric sites and can change 3D shape to change active site LO4