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MICR 271 FINAL EXAM QUESTIONS AND COMPLETE
ANSWERS
Replisome Speed - answer eukaryotic slower - must displace nucleosomes, cell-dependent regulation of replication multiple initiation sites to compensate Bacterial Replication Forks - answer 1. Helicase unwinds DNA, lagging strand coated with single stranded binding proteins, RNA primer synthesized by primase
- DNA Pol III holoenzyme tethered to DNA by beta clamp, loaded by t-clamp loader
- 1 Pol III core leading strand synthesis (continuous)
- 2 Pol III cores lagging strand synthesis (okazaki fragments)
- Pol I removes primer and fills with DNA, Ligase joins strands Eukaryotic Replication Fork - answer 1. Helicase (Mcm2-7) + proteins (GINSS, Cdc45, Mcm10) activate unwound DNA, ssDNA bound by replication protein A
- Helicase associates wtih primase (synthesizes RNA primer)
- Pol ε does leading, Pol δ does lagging (loaded onto DNA by proliferating cell nuclear antigen, carried to DNA by replication factor C)
- shorter okazaki fragments generation, Pol δ removes RNA primer and fills with DNA, ligase seals gap Family A DNA Polymerases - answer replication/repair Family B DNA Polymerases - answer DNA replication, 3'-5' exonuclease activity Family C DNA Polymerases - answer DNA replication Family D DNA Polymerases - answer DNA replication Family X DNA Polymerases - answer DNA damage repair Family Y DNA Polymerases - answer translesion DNA repair
Translesion Repair - answer insert bases opposite damaged nucleotides or repair dsDNA breaks Class I virus - answer dsDNA genome mRNA synthesis: transcription using host machinery, translatin mRNA --> proteins genome replication: using host machinery Class II virus - answer ssDNA genome (+ or -) mRNA synthesis: dsDNA transcribed into mRNA using host machinery genome replication: dsDNA produced and replicated, strands separated Class III virus - answer dsRNA genome mRNA synthesis: separated into ssRNA, + strand used as mRNA, translated into viral proteins genome replication: + ssRNA = template to produce new dsRNA Class IV virus - answer + ssRNA genome mRNA synthesis: + ssRNA used as mRNA, translated into proteins genome replication: + ssRNA used to produce - ssRNA = template to produce new + ssRNA Class V virus - answer - ssRNA genome mRNA synthesis: -ssRNA template to make + ssRNA (mRNA) translated into proteins genome replication: -ssRNA used to produce + ssRNA = template to produce new -ssRNA Class VI viruses - answer Retroviruses ssDNA reverse transcribed from RNA genome, dsDNA made using DNA Pol mRNA synthesis: dsDNA integrated into host genome --> mRNA --> protein genome replication: + ssRNA --> ssDNA by viral reverse transcriptase --> dsDNA into host genome by integrase --> +ssRNA to produce new genomes Class VII viruses - answer Reverse transcribing dsDNA genome mRNA synthesis: dsDNA --> mRNA by viral RNA Pol --> proteins
- DLP genome transcribed by viral RNA dependent RNA polymerase
- 11 dsRNA fragments genome transcribed individually by Pol complexes
- some ssRNA products associate 2nonstructural proteins and move to viroplasm
- 1 copy each genome segment incorporated into viral core
- virion structure assembled as moves out of viroplasm into ER Neuraminidase (NA) - answer cleaves sialic acid groups from glycoproteins, required for influenza replication Hermagglutinin (HA) - answer glycoprotein on influenza surface binds virus to cells with sialic acid on membrane Influenza Virus - answer -ssRNA genome
- virus HA attaches host receptors containing sialic acid, taken up by receptor mediated endocytosis (endoscope acidification stimulates virus uncoating)
- ssRNA containing polymerase complex migrates to nucleus
- replication -ssRNA =
- +cRNA: used to generate new progency RNA
- small viral RNAs: determines if RNAs used for transcription or replication
- viral mRNAs: used for translation after export to cytoplasm
- new viral RNA targeted for packaging by proteins HIV - answer Retrovirus
- tRNA hybridize PBS, rvs transcriptase fills PBS region
- RT synthesizes DNA complementary to U5 and R regions, alter used as primer
- RNAse H domain on RT degrades 5' RNA, removing U5 and R regions
- small - DNA primer moves to 3' end viral genome, hybridizes to R region on RNA
- strand DNA (cDNA) extended by RT
- majority viral RNA degraded by RNAse H domain of RT, leaving PPT region intact
- PPT RNA primes second strand synthesis
- second strand (DNA) displaced, complementary PBS regions anneal
- allows single stranded regions to be filled in, creating viral DNA with similar ends
- ~ 7 nucleotides synthesized, release sigma factor - elongation until termination
Factor Independent Release: mRNA forms stem-loop structure, RNA Pol falls off Factor Dependent Release: Pol pauses at end of coding region, interacts with Rho factor causes Pol to fall off Polyribosomal Complex - answer structure formed when coupling transcription + translation only in bacteria (no nucleus) Protein Synthesis - Initiation - answer 1. initiation factors (IFs), GTP, fMet-tRNA^fMet and 30S subunit assembles with mRNA
- 50S subunit joins --> IF2 hydrolyzes GTp, IF1 and IF3 released = 70S initiation complex Elongation Factor Tu (EF-Tu) - answer brings amino acid loaded tRNAs through ribosome Elongation Factor G (EF-G) - answer helps move tRNA and mRNA through ribosome Transcriptional Control of Gene Expression - answer control which genes transcribed or transcription rate
e.g. nucleoid associated proteins enhance or prevent access to DNA Post-Transcriptional Control of Gene Expression - answer control through mRNA modifications e.g. splicing in eukaryotes Translational Control of Gene Expression - answer control frequency and speed mRNA transcripts translated at e.g. secondary structure in 3' mRNA prevents transcript degradation e.g. small RNA binding to mRNA affects translation initiation Post Translational Control of Gene Expression - answer contorl rate protein becomes active/funcitonal e.g. phosphorylation, dephosphorylation Agonist: binds and activates receptor Antagonist: binds and blocks receptor Class I Bacterial Transcriptional Activator - answer activator protein associates upstream
- RNA Pol containing sigmaE transcribes genes for proteins to help cell deal with low pH
- low pH impacts cytoplasmic functions
- sigmaE bound RNA Pol increases transcription sigmaH, which upregulates transcription of genes to deal with cytoplasmic stress
- sigmaH upregulates genes for protein that enhances sigmaS translation
- sigmaS directions transcription genes to deal with low nutrient levels Prokaryotic Translational Regulation by microRNA - answer microRNA's = noncoding sectoin mRNA that folds into complex structure microRNA + Hfq protein bind target mRNA Outcomes:
- inhibit translation, stimulate mRNA degradation
- enhance translation (by competing with secondary mRNA structure that prevent translation)
- prevent 3'-5' exonuclease activity Eukaryotic Translational Regulation by microRNA - answer microRNAs = processed double stranded 22 bp mRNA hairpin loops
microRNA + RISC protein, helps present to target mRNA Outcomes:
- microRNA binds mismatched regions to target mRNA = translation inhibited
- microRNA binds precisely = stimulates mRNA degradation Translational Regulation by Hfq activity - answer enhances expression sigmaS - prevents formation of secondary structure sigmaS transcript that prevents translation Post Translational Regulation - answer 1. changes protein stability
- regulates biochemical activity
- affects protein localizing or targeting
- affects protein signalling Kinase - answer catalyzes transfer of phosphate from ATP receiving protein Phosphatase - answer removes phosphate Point Mutation - answer single baseinserted, changed, ro deleted Deletion Mutation - answer part of chromosome or DNA sequence lost Rearrangement Mutation - answer change in order of DNA bases
Antibiotics targeting 30S subunit - answer antibiotic interaction sites clustered along path mRNA and tRNA interactions Antibiotics Targeting 50S subunit - answer most antibiotic interaction sites clustered at or near peptidyl-transferase centre (PTC), where peptide bond formation occurs Streptomycin and Tetracycline - answer interfere with tRNA delivery Chloramphenicol and Clindamycin - answer inhibit peptide bond formation by preventing correct amino acid positioning of aminoacylated tRNA's in PTC Erythromycin - answer inhibits elongation by interfering with aminoacyl translocation Aminogycosides - answer interfere with peptide elongation = misreading of mRNA truncated proteins Puromycin - answer inhibits terminatino Mechanisms of Resistance to Ribosome targeting antibiotics - answer 1. impaired drug efflux due to low membrane permeability
- active efflux of drug from cell
- mutation of ribosome
- modification of target lowers drug affinity for target
- overproduction of molecule that mimics target, lowering effective drug concentration so target unbound
- recruitment specialized protein that activley removes drug form target
- drug modification
- drug degradation Eukaryotic Signal Transduction - answer receptor binds ligand = activation receptor phosphorylation starts reaction chain ultimately influencing gene expression Bacterial Signal Transduction - answer ligand or agonist from environment crosses membrane to access DNA and influence gene expression Pseudomonas Infecrtion - answer 1. signals interact with GacS protein, activated by phosphorylation (GacS-P)
- GacS-P transfers P to GacA (GacA-P, enhances transcription of microRNAs RsmZ and RsmY)
- microRNAs control whether RsmA (transcriptioal regulator) binds target promoter regions
polycistronic Tat - answer binds stem-loop at 5' viral RNA, activates viral transcription by stimulating elongation starting at viral long terminal repeat Rev - answer transports mRNA encoding viral structural proteins form nucleus to cytoplasm, increasing rate proteins synthesized Translational Control HIV and other viruses - answer 1. multicistronic transcripts (optimal translational control with small genome)
- virus makes overwhelming amount mRNA (outcompetes host mRNA for ribosome binding) Bioluminscence - answer used for communication system to manipulate behaviour host organism can help it search for prey, deter predators, camouflauge Marine Viruses - answer high conc in shallow areas and coral reefs viral mediated lysis of hosts releases dissolved organic matter (DOM) and particulate organic matter (POM) so available for other microorganisms + coral Benthic compartment - answer lowest level of body of water, containing sediment surface
Rhizosphere - answer soil region directly influenced by root secretions + associated soil microorganisms Commensal Microbes - answer derive benefit without hurting or helping plant Pathogenic Microbes - answer damage plant by infection or production phytotoxic compounds Beneficial Microbes - answer promote plant growth or protection Kudza Bug - answer feed on capsules of organisms that will live in bug as symbiont when born, if not present goes looking suggests --> animal behaviour shapes symbiont acquisition Green Iguana - answer young iguanas eat soil and feces to tailor microbiota suggests --> animals may adjust microbiota at different life-history stages Bobtail Squit - answer ejects bioluminescent bacteria daily suggests --> animals can actively control symbiont populations Fruit Fly - answer diet specific microbiota influence mating preferences
Sessile - answer surface-attached Mixed Species Biofilm Formation - answer 1. commensal organisms form biofilm, AI secretions low for intraspecies communications
- bacteria increase = AI2 increase until sensed by pathogens, attracted to and incorporated into biofilm Biofilms in Aquatic Environment - answer biofilm = food source other organisms occurs on ship hull - impacts fuel efficiency, introduces new species into new environment Biofilms on medical devices - answer indwelling devices (e.g. catheters, stents) quickly colonized Streamers - answer biofilms that bridge obstacles and corners in non-uniform environment cause disruption of flow challenge for medical device use in non-sterile environment Flora at Birth - answer bacterial in placental tissue, umbilical cord, amniotic fluid, meconium, fetal membranes vaginal tract + mothers gut flora
oral + nasopharyngeal flora established within hours of birth, lower intestine within 1 day delivery (vaginal/C-sec), diet (breast milk/formula), gestational age, sanitation, antibiotic treatment influence flora Commensal Fungi - answer problematic when colonize new areas body --> new niche = high growth level = immune response skin: few species, many on feet internal: on mucosal surfaces, many types Normal Fungi - answer small component of commensal flora health contribution unknown normal fungi change more than normal microbiota (due to envirnoment) prolonged antibiotic treatment = predispose fungal infections Viruses as Commensals? - answer body can harbour persistent viral infection may protect or predispose person to infections
