Download STUDY GUIDE for Dr. Mohnen`s part of Exam #3

Study Guide for Mohnen portion of EXAM #3 BCMB/BIOL/CHEM 3100
(MWF, Tu breakout; 10;10-11:00 class) Drs. Debra Mohnen and Daniel DerVartanian
General list of material you are responsible for for Dr. Mohnen’s part of
Exam #3. This includes material on the last part of lipids; nucleic acids; DNA
replication; transcription and translation. [NOTE: Exam #3 will cover the last part of
the material covered by Dr. Mohnen and the material covered by Dr. DerVartanian. This
document is meant only as a guide for your preparation for that part of the material from Dr.
Mohnen.]
You are responsible for all material covered in the lecture and in the breakout sections, and in the
class text, as described by Dr. Mohnen, regarding lipids (relevant sections of Chapters 11, 12,
13) nucleic acids (Chapter 33) , DNA replication (Chapters 34-35), transcription (Chapters 3638) and translation (Chapters (39-40).
The topics covered include, but are not limited to the following.
The part of the material on lipids that we did not cover on Exam 2. This material starts
with the class notes on Lipid rafts and goes through the end of those notes (including the
material on these topics in the book). [This includes the following.]
Make-up and principals of biological membranes
Lipid rafts
Three types of membrane proteins (in detail): peripheral, integral and lipid-anchored (3 types)
Membrane transport: including passive, primary active and secondary active (and examples of
each, in detail)
Signal transduction: including G proteins, and detailed examples and understanding of different
signal transduction cascades including: common secondary messengers, seven transmembrane
helix receptors, adenylyl cycles pathway, inositol phospholipid signaling, receptor tyrosine
kinases
Structure of DNA and RNA
Know and be able to draw structures of 5 bases and their nucleosides and nucleotides for RNA
and DNA
Where H-bonding occurs between bases
Names and abbreviations of bases, nucleosides and nucleotides
Understand pyrimidines and purines and the preferred conformation in biological organisms
Nucleotides usually complexes in vivo with divalent cation (e.g. Mg++)
Nucleotides as building blocks of nucleic acids (process, i.e. nucleophilic attack of 3’-OH on αphosphate of incoming NTP
History of DNA: Friedrich Miescher, Fred Griffith, Watson and Crick, Avery, MacLeod &
McCarty, Roger Herriott & Alfred Hershey, Chargaff rule
DNA double helix: rise = 3.3 Å, pitch = 34 Å, 10.4 bp/turn, 1000 bp = kb
Major groove vs minor groove
Noncovalent forces that hold helix together
A vs B vs Z DNA
Different types and sizes of chromosomes
Melting point: Tm, melting curve, OD 260 of nucleic acids, ss vs ds DNA
DNA is supercoiled in vivo
Topoisomerases
RNA: rRNA, tRNA, mRNA, small RNA
RNAs involved in protein synthesis, post-transcriptional modification & DNA replication,
regulatory RNAs
Bacterial rRNA:5S+23s+16s; eukaryotic rRNA: 55s+5.8s+28s+18s
Hairpins
Chromosome packing: nucleosome (2x H2A, H2B, H3, H4) = histone octamers + 146 bp DNA +
H1 + 54 bp DNA
Packing of DNA: nucleosome (10x); solenoid (4x); protein scaffold (200x)
Nucleases: specify 5’ vs 3’ ends, endo vs exo, understand how to denote and read specificity of
cleavage
RNA is less stable than DNA, RNA sensitive to alkali hydrolysis
Restriction enzymes; be able to read and use restriction enzyme tables, blunt end vs stick end
Palindromes; use of restriction enzymes
DNA Replication: DNA Repair and Recombination
Semiconservative vs conservative DNA replication, Meselson & Stahl
Arthor Kornberg: discovery of DNA Pol I
Function of each of activities of DNA Pol I: 3 activities:
5’→3’ polymerase, 5’→3’ exonuclease; 3’→5’ exonuclease (and functions in vivo!)
Synthesis, editing/primer removal, proofreading
Klenow fragment
processivity
Delucia & Cairns experiment
DNA PolI vs DNA Pol III, and other DNA polymerases
DNA synthesis in E. coli
Initiation: OriC
Replisome: primosome (primase+helicase), DNA Pol III, ss DNA binding proteins
Okazaki fragment, leading vs lagging strand
DNA replication: Ori C, Terminator region (tre), nick translation, RNA primer, DNA ligase
Difference between prokaryotic and eukaryotic DNA synthesis
Telomers, telomerase
Sanger Sequencing: (dideoxy sequencing)
DNA repair: error rate of DNA synthesis by different enzymes and with repair enzymes
direct repair (photolyase) vs excision repair pathway (know pathway)
endonuclease/DNA glycosylase removes damages/deaminated bases and gap filled by
DNA Pol I
Types of DNA damage
PCR
RNA Synthesis & Regulation in Bacteria; Gene Expression in Eukaryotes; RNA
Processing in Eukaryotes
Gene: DNA that is transcribed
RNAs:
rRNA: 80% of RNA, part of ribosomes
tRNA: 15% or RNA, 73-95 nucleotides long
mRNA: discovered by Jacob & Monod, 1-3% of RNA, unstable
small RNA: may have catalytic activity (e.g. U6 & U2 of spliceosome); sn RNA (part of
splicing machinery), snoRNA( rRNA biogenesis and modification),
microRNA (regulates use of RNA)
Small interfering RNA (siRNA): antiviral defense mRNA degradation
long non-coding RNA (lncRNA): gene regulation
Piwi interacting RNA (piRNA): gene regulation
RNAs involved in protein synthesis, post-transcriptional modification & DNA replication,
regulatory RNAs
E. coli RNA Pol holoenzyme (α2ββ’ωσ) vs core enzyme (α2ββ’ω)
E. coli RNA Pol does all three: initiation, elongation, termination
Searches for and finds initiation sites (~2000 in E coli), unwinds DNA, finds
transcription start
site, totally processive, detects termination signals, interacts with activators & repressors
σ recognizes promoter; σ70 recognizes promoters of housekeeping genes
Consensus sequences in E. coli: TATA box (TATAAT); -35 region (TTGACA)
Strong vs weak promoters
Coding strand vs template strand and relationship to mRNA
Initiation of transcription: holoRNA Pol
Elongation: δ leaves and NusA binds
Termination: RNA hairpin causes strong pause, some times Rho protein (6mer with ATPase
activity) involved
Eukaryote RNA Pols:
RNA Pol I: rRNA, 18s, 5.8s, 28s
RNA Pol II: mRNA, snRNAs (U1, U2, U4, U5)
RNA Pol III: t RNA, 5s rRNA, U6 snRNA, small RNA
Effect of α-amanitin on different RNA Pols
Regulation of housekeeping vs differentially expressed genes
Transcription factors: activators vs repressors (both can be allosterically regulated)
Repressor: inducer vs corepressor
Lac Operon: lacZ (β-galactosidase), lacY (Lactose permease), LacA (thiogalactoside
trasnacetylase)
Operator region in Lac Operon
Lactose vs allolactose, escape synthesis vs induced synthesis
Catabolic repression: concept that in presence of Glc PEP-dep sugar phosphotransferase system
causes Glc to be phosphorylated to Glc-P
However, in absence of Glc, P is transferred to Adenylate cyclase yielding cAMP which
activates cAMP regulator protein (CRP) (also called catabolite activator protein, CAP)
yields ~50X stimulation of transcription at the Lac operon
Eukaryote promoter elements:
RNA Pol II promoters:
enhancer + TATA box (-24 to-32) + Inr (-3 to +5)
enhancer + Inr (-3 to +5) + DPE (+28 to +32)
Inr: initiator element; DPE: downstream core promoter element
Cis-acting element: DNA sequences that regulate expression of gene located on same DNA
molecule
Transcription initiation in eukaryotes:
TFII: transcription factor for RNA Pol II (TF-D (with TBP),A,B,F (then initiate),E,H
TFIIH: opens double helix & phosphorylated CTD of RNA PolII change from initiation to
elongation
Enhancer: cis-acting element that can be on either DNA strand and stimulate transcription even
1000s of bp away
Most eukaryote TFs interact with multiple proteins to give large complexes
Mediator: complex of 25-30 subunits interact with transcription machinery before initiation
begins; bridge between enhancers and promoter-bound Pol II
Steroid Hormones: estradiol as example
Estrogen receptor is nuclear hormone receptor; has DNA binding domain (with zinc-finger
domain) and ligand binding domain
Binding of estrogen receptor with estrogen allows coactivator to bind nuclear hormone receptor
that is bound to DNA of specific genes
Coactivator: protein that binds to receptor only after is has bound ligand; coactivators can
stimulate transcription by loosening interaction between histones and DNA, making DNA
more accessible to transcriptional machinery
Histone acetyltransferases (HATS) acetylate histones; this reduces affinity of histones for DNA
and generates docking site for transcription factors that have Bromodomains (domains that
bind to acetylated histones and acetyllysine)
Bromodomains are present in chromatin-remodeling machines (ATP-powered complexes that
make DNA in chromatin more accessible)
Histone deacetylases can remove acetyl groups resulting in inhibition of transcription
Processing of RNA: cleavage, modification of nucleotides, addition of nucleotides
Both prokaryote and eukaryote rRNA transcripts contain multiple rRNA and tRNA genes that
must be removed from the primary transcript by endonuclease action
Eukaryote pre-rRNA is first undergoes nucleotide modification prior to cleavage of the
individual 18s, 5.8s and 28S rRNA
For RNA Pol III: RNaseP (ribonucleoprotein, 377 nucleotide RNA + 130 kd protein) remove
nucleotides from the 5’ end of the rRNA precursors and CCA-adding enzyme adds
nucleotides to 3’ end of tRNA; 30% of nucleotides in tRNA are modified
In prokaryotes mRNA is NOT further processes. Transcript is DIRECTLY translated.
In eukaryotes:
mRNA: 5’ capping: 7-methylguanylated-5ppp-5’ cap
3’ poly adenylation: Add ≤250 adenylates, AAUAAA polyadenylation signal
CPSF: cleavage & polyadenylation specificity factor bind endonuclease and polyA
polymerase adds AAAAAs
Splicing: takes place on spliceosome, complex of ≥100 proteins & 5 snRNAs (associate with
proteins to give snRNPs)
Vertebrate splice site: exon-GU………A…….AG-exon
U1 at 5’ splice site, U2 at branch point, U5 at 3’ splice site + U4/U6
!! U6 snRNA and U2 sn RNA form catalytic center of spliceosome!, U4 masks U6 until splice
sites are aligned
Yields lariat and spliced exons
Alternative splicing: membrane vs soluble antibody
CTD of RNA Pol II recruits enzymes to synthesis 5’ cap, splicing complex, and endonuclease for
poly A tail generation
RNA editing: apolipoprotein B in liver vs small intestine; post transcription modification of C to
U introduces a stop codon
Ribozymes: self splicing RNA, some introns (group 1 introns)
The Genetic Code; Mechanism of Protein Synthesis
Genetic code: 3 base code, sequential, non-overlapping, degenerate
2 letter: 42 = 16; 3 letter 43 = 64; 4 letter 44 = 254
Adapter molecular hypothesis: Crick, 1958
Codon, reading frame, AUG: start codon; stop codons (UGA, UAG, UAA)
tRNA: anticodon, 3’ end is where amino acid is added, 30% nucleotides in tRNA are modified
wobble in base-pairing of 5’ anticodon (== 3’ in codon in mRNA)
ALL 3’ends of tRNAs end with 5’-…….CCA-3’
aatRNA = activated tRNA
aa + ATP = AMP=aa + PiPi
aa-AMP + tRNA = aa-tRNA + AMP
Aminoacyl tRNA synthetase catalyzes reaction
Some Aminoacyl tRNA synthetases can edit wrong aa: Threonyl-tRNA has CCA are that can
swing and remove Ser if incorrectly inserted
Ribosomes = RNA + proteins
Translation: mRNA read 5’ to 3’; protein synthesized in N to C terminus direction
Prokaryotic ribosomes: 30S = 21 proteins + 16S rRNA; 50s = 31 proteins + 23S rRNA +
5S rRNA; 30s+50s=70s
Eukaryotic ribosomes: 40S = 30 proteins + 18S rRNA; 60 S = 40 proteins + 28S rRNA + 5.8S
rRNA + 5S rRNA
40S+60S = 80S
Ribosome: three sites – A site and P site and E site
RNA is catalyst in ribosome!! Peptidyltransferase activity in 23S rRNA of 30S subunit!
Polysomes
A site for all aa-tRNA except initiator tRNA
P site for all peptidyl tRNA except also for initiator tRNA
Eukarytote: first AUG is start codon
Prokaryote: first AUG downstream of Shine-Dalgarno sequence (purine rich region) is start site
1st AUG recognized by initiator tRNA (in prokaryote this tRNA carries formylMet), in
eukaryotes this is Met
3’ end of 16S rRNA in 30S ribosome subunit binds to Shine-Dalgarno sequence
30S + IF1+IF3+IF2GTP +fMet-tRNA binds mRNA
THEN 50S binds, IF1, 3, 2 released and 70S ribosome with mRNA and initiator tRNA aligned at
correct position is assembled.
EF-TU-GTP brings in each aatRNA to ribosome in A site, using GTP
EF-TU-GDP interacts with EF-Ts to lose GDP and bind GTP for next escort function
Α-NH2 group of aa makes nucleophilic attack on carbonyl C of ester linking aa to tRNA this
transfers growing chains from tRNA in P site to tRNA in A site
Peptidyl tRNA in A sites moves to P site via aid of EF-G (translocation) costs GTP
Termination: RF1 (UAA & UAG), RF2 (UAA & UGA), RF3 (binds GTP) causes stalling of
ribosome
Cleavage of 4 phosphoanhydride bounds:
ATP→AMP for aa tRNA production (note: ppI → 2 Pi)
GTP in elongation (EF-TU –GTP)
GTP in translocation (EF-G-GTP)
mRNA in eukaryotes is circular because of interactions between proteins that bind the 5’ cap and
those that bind the poly A tail
The following were not discussed in detail in class in 2015, and you are not responsible for these
on Exam #3 in 2015. None-the-less, the concepts are important.
Post translational modification
Co-translational & post: deformylation of formyl Met, removal of N terminal Met, disulfide
bonds, cleavage of proteins, phosphorylation, glycosylation, acetylation
Eukaryotes:
Signal hypothesis: N-terminal 16-30 aa residues, highly hydrophobic
Translation of signal peptide → N-terminus binds to SRP (signal recognition particle) =
protein/RNA complex (7 SL RNA + 4 proteins)
Binds to SRP receptor (docking protein) on ER
Anchored by ribophorins (ribose-binding protein)
Signal peptide cleaved by signal peptidase
Glycosylation: start in ER finish in Golgi