BMED 3600 Spring 2015

Exam 1 Review Sheet

Topics to focus on:

- Introduction to cells:
o Differences between prokaryotic and eukaryotic cells
 Prokaryotic
 2 cell membranes
 Cell wall
 No organelles
 No nucleus
 Eukaryotic
 1 cell membrane
 No cell wall
 Organelles
 Membrane bound nucleus
o Cell anatomy
 Cell membrane
 Phospholipid bilayer
 Fluid
 Proteins
o Integral (signaling/structure)
o Surface (signaling/structure)
o Transport (usually integral
 Nucleus
 Houses DNA
 Porous
 Inner and outer membrane (nuclear envelope)
 Organelles
 ER
o Rough = protein synthesis
o Smooth = lipid synthesis
 Golgi apparatus
o Works with ER
o Packaging
 Mitochondria
o 2 membranes
 Inner is folded
o Matrix (inside inner membrane) contains oxidation enzymes
o ATP formation
o “powerhouse”
o Self replicating (has its own DNA)
 Lysosomes
o Intracellular digestion
  Peroxisomes
o Self-replicating
o oxidases

- Proteins:
o Levels of structure
 Primary: amino acid sequence
 Secondary: folding sequences
 Alpha helix (hydrogen bonding)
 Beta sheets
 Tertiary: over all 3D shapes
 domains
 Quaternary: different polypeptides bound together
 Creates binding sites
 Protein subunits
 Dimers and tetramers
o Protein stability
 Hydrogen bonding between side chains of amino acids
 S-S bonds
o Protein regulation
 Allosteric enzymes
 Binding sites that are separate from the ligand binding site and turn the
enzyme on or off
 Side chain modification
 Phosphorylation to turn proteins on or off
o Kinases phosphorylate
o Phosphotates dephosphorylate
 GTPases: GTP binding sites
 GTP = active
 GDP = inactive
 GAP dephosphorylates
 GEF removes bound GDP which is replaces by GTP (activates)
- DNA & chromosome structure
o DNA structure
 Double helix
 Anti-parallel, complementary polymer strands
 Hydrogen bonding between nucleotide base pairs
 G&C
 A&T
 Minor and Major groves
 Mostly right handed
 Deoxyribose = sugar
 Phosphate bonded to 3’ carbon
 Phosphodiester bond between phosphate on one sugar and 5’ carbon of
another sugar
 o Different chromosome structures
 Very loose during interphase (replication)
 Referred to as “chromatin”
 “beads on a string”
o 1 bead and following string = nucleosome
o Bead = histone core
 H1, H2A, H2B, H3, H4
 Nucleosomes wrap into
o Zig zag structures
o Solenoid structures
o 30nm
 Mitosis: condensed, different strands are separated into sister chromatids
 Chromatin folds a lot a lot a lot to become tightly packed with two sister
chromatids joined at a centromere
o How is DNA packaged into different chromosome structures
 H1 histone binds to nucleosome chore and can dictate what direction
the leaving DNA points
 Winds and unwinds rapidly
 Chromatin-remodeling complexes
o Large numbers
o ATP dependent
o ATP hydrolysis spins DNA around histone to expose new
sections
- DNA replication
o DNA replication machinery components
 Helicase
 Unwinds DNA
 Polymerase
 Adds nucleotides via “templated polymerization”
 Always 5’ -> 3’
 Catalyzes phosphodiester bond
 RNA primase
 Adds RNA primer for beginning of leading strand and okazaki fragments
 Ligase
 Replaces primers and facilitates phosphodiester bonds of okazaki
fragments
o DNA replication of leading and lagging strands
 Happens in 2 directions (beginning at origin of replication)
 Initiator proteins pry bases apart
o A and T easier to pry apart so usually start in areas rich in those
 Sequences mark areas of replication origin (promoter)
 Helicase creates a replication fork in 2 directions
o 4 sites of replication
 Polymerase is attracted to the area
 Leading strand
 Replication happens continually
 Only one primer
  “follows” helicase
 Lagging strand
 Replication happens in sections
o Clamp dissociates and polymerase unattached from DNA when
it reaches an RNA primer
 Many primers
o Primase activated as helicase continues to unwind
o Single strand binding proteins protects DNA until polymerase
comes back
 Sections called Okazaki fragments
o Polymerase continues replication after the primer
o Repeat from beginning
 Ligase connects everything
o How are the ends of chromosomes replicated
 Telomerase replicates the end of chromosomes
 Uses RNA template attached to enzyme to at telomeres
 Short repeats in DNA sequence
 DNA polymerase(alpha) completes complementary strand

- DNA repair and recombination

o That mechanisms are used to fix different errors
 Proofreading
 “fingers” of polymerase must tighten and do so more readily with
correct nucleic acid
 DNA polymerase needs 3’-OH group to continue replication
 Incorrect nucleotide does not hydrogen bond and does not free up the
3’-OH group for elongation
 Exonuclease on polymerase cuts off nucleotides until it sees a 3’-OH
group
 Elongation continues
 Base Excision Repair
 DNA glyoclysase removes base only
o Deaminated C, depurinated A or G, alkylated or oxidized bases
o “flips out” bases to check them
 AP endonuclease removes the sugar group
 DNA polymerase adds the correct nucleotide
 Ligase facilitates phosphodiester bond
 Nucleotide Excision Repair
 Repairs large changes in structure
o Pyrimidine dimers
o Covalent reactions with large hydrocarbons
 Scans DNA (multiprotein complex)
 Nuclease cuts phosphodiester bonds around damaged section
 Helicase takes away damaged section
 Polymerase and ligase re-add undamaged section
  Double strand break
 Really bad; could end up losing sections of genes
o Reactive metabolites
o Chemical exposure
o radiation
 Non-homologous end joining
o Loss of nucleotides due to degradation of ends
o Broken ends brought together
o Rejoined by ligase
o Loss of nucleotides
 Homologous recombination
o Loss of nucleotides due to degradation of ends
o Exonuclease degrades 5’ end
o Strand invasion with sister chromatid
 Heteroduplex holds molecules together by crossing over
o DNA replicates according to sister chromatid (both sides)
o Ligase fixes phosphodiester bond gaps
o Also happens during meiosis for more genetic variability
 Cleaves holliday junctions (by heteroduplex)
 Cleavage of template and complement causes
crossover
 Ligase connects pieces from sister chromatids

- Transcription
o Different kinds of RNA
 mRNA (proteins)
 rRNA (core of ribosomes)
 tRNA (carry amino acids to ribosomes)
 snRNA (cut pre-mRNA into mRNA)
 snoRNA (modifies rRNA)
 scaRNA (modifies snRNA and snRNA)
 miRNA (blocks transmission of mRNA)
 siRNA (degrades mRNA)
 other (telomere synthesis, protein transport in cells)
o Basics
 Initiation
 RNA polymerase binds to promotor (factors needed)
 Separate DNA strands
 Template strand enters active site
 Elongation
 Dissociates from promotor
 Adds ribonucleic acids
 Termination
 Pre-mRNA released from RNA polymerase
 Polymerase removed from DNA
 o Prokaryotic Transcription
 Sigma factor binds to promotor and RNA polymerase
 Leaves after promotor
o Transcription in eukaryotes vs prokaryotes
 Eukaryotes require transcription factors
 Eukaryotes have complex DNA packaging
 RNA processing
 Initiation in Eukaryotes
 TFIID binds to TATA box through TBP
 Binding of TFIIB/H/F, RNA polymerase II, and others
 TFIIH has helicase and kinase activity
 Polymerase disengages TFs
o mRNA processing/maturation
 RNA capping
 Modified guanine
 polyadenylation
 RNA splicing (splicesome)
 Removes introns
o Noncoding
 Leave exons
o Coding
 One gene can make multiple proteins
 3 splice juntions
o Splice donor: 5’ end of intron (exon-GU)
o Splice acceptor: 3’ end of intron (AG-exon)
o Branch site: atleast 1 A, 30 nts up
 Cut at donor, bond to branch site (lariat), cut at acceptor

- Translation
o Key players in translation
 tRNA
 adapter
 contains anticodon that binds to codon and drops of amino acid
 wobble base pairing
o only requires accurate matching of first 2 nucleotides instead of
all 3
 aminoacyl-tRNA-synthetases: couple amino acids to tRNA; 1 for each amino acid
 Ribosomes
 Catalytic machine
 Small unit: tRNA can be matched to codons
 Large unit: catalyzes peptide bond
o Steps involved in translation initiation
 Recognize start codon (AUG)
 Attaches methionine
 Has to be tRNAimet to start, otherwise tRNAmet just adds methionine
 Pre initiation complex
 40s and eIF3 interact with eF1A
  Met-tRNAimet and eIF2-GTP
 Initiation complex
 eIF4 complex binds 5’ cap of mRNA to 40s subunit at eIF3
 eIF3B removes secondary factors (helicase component)
 Scanning for start codon
 Hydrolysis of eIF2-GTP prevents further scanning once codon is found
 Formation of 80s ribosome
 eIF 1,2,3, and 4 dissociate
 eIF5 and 6 catalyze large subunit binding to small subunit
o hydrolysis of GTP on eIF5 (proof reads)
o Steps involved in translation elongation
 eF1A and GTP bring tRNA to A site
 when correct anticodon, GTP hydrolyzed and eF1A-GDP dissociate
 conformational change
 peptide bond formation: large subunit catalyzes
 hydrolysis of EF2-GTP moves tRNA from A site to P site
 tRNA at E site (after P) leaves when A site is filled
 eRF1 reconizes stop codon in A site
 eRF2 (GTP binding) cleaves polypeptide from tRNA
o Fate of mis-folded proteins
 Molecular chaperones: ATP hydrolysis
 Helps fold correctly
 Recognizes mis-folds
o Hydrophobic groups on outside instead of inside
 Halt mis-folding
 Re-folding
 Proteasomes
 Recognize proteins marked with ubiquitin
 Unfold proteins
 Destroys as threaded through proteasome core
- Control of gene expression
o Purpose of gene expression control
 Differentiate cells without losing genetic information from cell to cell
o Different methods used to control gene expression (the basics, the major players)
 Modification of chromatin
 Chromatin remodeling complex
 Histone chaperones (remove)
 Histone modifying enzyme
 Transcription control proteins
 See operons
 Mediators (eukaryotic cells only) regulatory proteins affect places far away
 Promotors (bind to transcription factors and polymerase)
 Enhancers (bind to regulatory proteins)
 Insulators (block enhancers)
 Repressors also bind to activators in eukaryotes
 Alternative splicing
 Regulated nuclear transport
  miRNA/siRNA
 Ubiquitin
o Fundamentals of how operons work
 Collection of genes used in one mRNA strand
 Negative control, ex: Trp operon
o repressor protein binds to DNA when protein is in high
concentration -> turns off gene
 Positive control, ex: CAP in E.coli
o Activator helps RNA polymerase bind, high concentration of
ligand removes activator -> turns gene off
 Combinations: Lac operon
o Low glucose turns on activator, high lactose turns off repressor
-> turns on gene (digest lactose instead of glucose)
o Techniques used in gene expression control discovery
 Random cell’s nucleus could be used to clone things so must hold entire
genome
o Fundamentals of how siRNA and miRNA work
 Bind to complementary mRNA strands and either degrade or sequester
 miRNA can sequester
 siRNA can only degrade
 dsRNA gets diced up via Dicer to make siRNA
 siRNA binds to RISC to destroy unwanted mRNA