Test #2 Flashcards
Types of Genomes
Genomes can be Single Stranded DNA, Double Stranded DNA, Single Stranded RNA, Double Stranded RNA
RetroVirus
Virus with an RNA Genome
BAC Cloning
Computer searches for common sequences in DNA by finding tagged sites or sequences. These are found in fragments. Computer finds overlaps and combines them to map genome
Synteny
Similar genes in a similar pattern among different species
Introns and Extrons
Introns: non coded regions in between coded regions.
Exons: the expressed genes
Alternative splicing
Removal of Introns and adding Exons together to form multiple proteins made from one gene
Human Genome DNA (Protein)
Only 1.5% of DNA codes for proteins
Genomic Alterations
Gene duplication: Duplication of gene next to it on the same chromosome
Transposition: moving a gene from one spot on the chromosome to another
Inversion: ???
Single Nucleotide Polymorphism
Single base variation in certain areas of the genome that are different across different people.
Haplotype
set of SNP’s
Linkage Analysis
Mapping heritable trait genes to their chromosome locations. Can examine inheritance pattern of DNA markers within families to determine if there is a relationship between a particular region of genome and phenotype
First Dimension Gel Electrophoresis
Proteins are separated according to isoelectric point
2D Dimension Gel Electrophoresis
SDS-Page to separate protein according to size. Used to compare two or more different samples (Cancer vs. no cancer) to identify proteins expressed
Supercoiling
Tight coiling of DNA for DNA packing and regulation
Supercoiling effect on transcription and replication
strand separation leads to added stress and super-coiling. As a result, the DNA becomes over-wound ahead of the polymerase, and under-wound behind.
Topoisomerase I
Relaxes negative supercoils.
Chancges linking # by 1 in positive direction. Nicking 1 stand and passing unbroken strand through the break
Topoisomerase II
DNA Gyrase
Introduces Negative supercoils, needs ATP. Changes linking # by 2 in negative direction. One intact duplex DNA segment passes through a double-strand break in another segment (breaks 2 strands)
Topoisomerase IV
Resolves Catenanes that arise in DNA replication. Passing one duplex thorough a double strand break. No ATP Required
Catenanes
Intertwined Bacterial DNA because of replication
Cohesins
Bind to Chromosomes during G1 Phase. Keep sister chromatids together during S phase DNA replication until anaphase
Condensins
Bind during Mitosis and keep chromatids condensed until separation during anaphase
SMC Proteins
Structural Maintenance of Chromosomes. Include Cohesins and Condensins. Homodimers in Bacteria. Heterodimers in eukaryotes
Histone core (histone octamer)
made of 2 sets of H2A, H2B, H4 and H3.
Histone Proteins
DNA wraps around nearly twice for each histone octamer. Histone has Lysine and Arginine so it is positively charged.
H1
Completely blocks gene expression by locking in the nucleosome
Chromatin Remodeling Complexes
Can move a histone by reposition. Eject a Histone.
Or replace Histones with altered histone for different interactions.
Acetylation:
Phosphorylation:
Methylation:
Neutralizes Charge
Decreases charge
NO change in charge
Epigenetics
During replication H3-H4 are distributed between old strand and New strand (every-other to one). H2A-H2B are then added to make full octamer. Epigentic markers fill in gaps with new histones that match original.
DNA replication method
Semi-conservatively. always goes 5’—>3’. Needs a free OH on the 3’ for proper replication. Replication is bidirectional
Replication Fork
Replication is coordinated in both direction 5’-3’ Lagging strand creates Okazaki Fragments. Continuous strand continually goes
DNA Polymerase
Needs template strand, Synthesizes in the 5’-3’, Many have 3’-5’ proofreading exonuclease activity to back up and fix something.
Polymerase I
Okazaki fragment Processing and DNA repair. 3’-5’ and 5’-3’
Polymerase III
Chromosome replication. 3’-5’ exonuclease
Enzymes required at the DNA replication Fork
Beta clamp, Helicase, Topoisomerase, DNA Primase, Ligase, SSB
Polymerase II
Transleasion repair. It has a 3’—5’ exonuclease
Polymerase IV
Transleasion synthesis . It has no exonuclease ability
Polymerase V
Transleasion synthesis. It has no exonuclease ability
Processivity
number of nucleotide that a polymerase can incorporate into DNA during a template-binding event before dissociation from the DNA. (B Clamp increases processivity by keeping polymerase on DNA)
How can we take samples every second
Mg2+ is needed for the DNA polymerase, Adding a chelating agent can stop the binding of Mg2+ and thus stop the reaction.
DAM methylase
adds methyl groups to the origin of replication. Adds after replication begins to regulate only one DNA process.
DnaA-ATP
Promotes an open complex in chromosome, closes when losing a phosphate group. (E.Coli)
Tus/Ter sites
Tus proteins bind to Ter sites
Tus Ter system prevents a replication fork from extending much beyond the halfway point around the chromosome. ensures that the fork moves in the same direction as transcription.
ORC
Origin recognition complex (eukaryotic)
Telomeres
buffer at the end of eukaryotic chromosomes. Each time replication happens, small part of the telomere is left out.
Also form T-loop to protect the chromosomes from nucleases
DnaA
initiator , binds oriC (Prokaryotic)
HU
Stimulates open complex at oriC (Prokaryotic)
DnaC
Helicase loader
DnaB
Helicase
Gyrase
Type II topoisomerase (Prokaryotic)
SSB
Stabilizes and protects single stranded DNA form nucleases
Primase
synthesizes lagging-strand RNA primers
SeqA
Binds hemimethylated GATC sequences
Hda
induces DnaA to hydrolyze ATP
silent mutation
change in DNA but no change in amino acids
Nonsense
new codon calls for stop codon. Half a protein made or small portion
Missense
Change in amino acid
Conservative: new amino acid is very similar to old so didn’t make a difference.
Non-conservative: new amino acid is nothing like original.
Transition
One purine replaces another. (or pyridine replaces pyridine)
Transversion
a pyrimidine is replaced with a purine or vice versa
Frame shift.
Brought about by deletion or insertion of base pairs. deletion doesn’t do much if deletes by sets of 3. Often leads to premature stopping (nonsense)
bacteria mismatch repair
Mut proteins distinguish the mother and daughter strands by methylation patterns. MutS and MutL create a dimer and scan for methylated groupd, ONly daughter is non methylated GATC.
Helicase II nicks at the GATC site and allows dna.
Deamination
loss of an amine group, caused by water or nitrous acid. ( C –> U or C –> T)
Depurination
loss of purine based caused by water
ROS ( reactive oxygen species
can add oxygens to bases ,==
Base Excision Repair
Glycosylase flips out wrong base from DNA Strand. AP Endonuclease can knick at that base and cut that whole section of the strand out.
DNA pol I can then fill the gaps.
Methyl Transferase
removes unwanted from bases. Can only be used once, then it grandness
UV light efffects
Crateas a covalent bond on the same side to form thyminedimer.. The DNA is then bent and cant’ be replicated
Photorepair
Only done by prokaryotic. DNA photolyase can use light energy to break the covalent bonds between the dimers
Nucleotide excision Repair
1- 2 UvrA and a single UvrB bind at site of dimer.
2- UvrA leave and UvrC is recruited. UvrC 5’ and 3’ cut the DNA out of double strand on both sides of dimer spot. 3- Polymerase I can now re-synthesize DNA from template, Ligase seals nick.
these are bacterial enzymes
humans use XP proteins.
How to avoid lesion
1) translesion synthesis: last resort. Polymerase IV or V just throw in a random base.
2) Fork stalls: fork regression, good strand continues to replicate and can then flip back and be used as template for strand stuck at lesion.
3) Recombination repair
Gap Repair
Recombination ( switchin spots of DNA strands) so that the newly made daugter strand can act as a template for the stalled strand.
Double strand repair steps
1- Nucleases chew away 5’ end to make 3’ overhang
2- single strand overhang invades the complementary region in the intact homologous chromosome.
3- Other overhang also invades
4- Use the homologous sequence as a template to repair the gap.
Two possible products for holiday intermediate/double strand break repair
Non-crossover: ends are still the same. (X-X cut)
Crossover: different chromosomes have swapped chunks of the chromosome. (X-Y cut)
RecBCD helicase/nuclease
Required for processing double-stranded breaks in the DNA before recombination can take place. Makes the 3’ end extension/overhang.
RecA
Requires ATP. Displces SSB grows along DNA 5’–3’ and protects the 3’ end extension
RuvAB
Binds DNA and promotes branch migration for recombination
RuvC
Resolves Holiday intermediate by cutting DNA back into two separate molecules.
Spo11
catalyzes and processes double stranded break recombination ONLY during Meiosis. Spo11 breaks both strands of DNA through a covalent attachment to DNA through the tyrosine amino acid in the active site.
Non-homologous End Joining
Last resort. Forces annealing of double stranded breaks
RNA pol vs DNA pol
Both are 5’-3’ but RNA does not need a primer.
RNA Polymerase (bacterial core)
Consists of 2αββ’(ω). Bacteria only have one RNA polymerase, gaining specificity through sigma factors.
Consensus Sequence of Bacteria
-35: TTGACA
-10: TATAAT
These are the primary housekeeping genes
Promoter doesn’t have to perfectly match for transcription but closer to consensus sequence, more and faster production
Termination of Transcription
RHO-independent: Hairpin forms on the RNA transcript due to base pairing, followed by a long string of UUU’s
RHO-Dependent: Rho-helicase runs along the RNA transcript. Catches up to the RNA polyermase, then pushes pol off
RNA Polymerase I
185, 25S and 5.85 rRNAs
RNA Polymerase II
mRNA, microRNA some non-coding RNA
RNA Polymerase III
tRNA, 5S rRNA, 7SL RNA
RNA Polymerase proof reading
RNA pol can remove several bases to return back to a its original mess up. Long spread gives reach to several base pairs and several opportunities to catch mistakes.
DNA Footprinting
DNA is continually eaten away by DNase enzyme. Where protein is bound, it will not but cut and will not produce strands.
Steps at which regulation occurs
Transcription initiation, RNA processing, RNA stability, Protein synthesis, Protein modification, Protein transport, Protein degradation
Negative control
repressor binds to DNA to shut down transcription. Can be activated or deactivated by effectors.
Positive control
Activator binds to DNA to turn on transcription. Can be activated or deactivated by effectors
Looping
if the enhancer is too close to the promoter, an architectural regulator can drastically bend DNA for proper contact. Various other ways, think of them too.
Coactivator and Corepressor
act as bridges between other proteins, such as activators and polymerase.
Insulator Proteins
Binds to an insulator site/sequence to keep regulators and promoters form one gene separate from another gene.
Combinational regulation
various transcription factors that can have multiple binding sites for one gene. This allows more regulation on genes and creates a spectrum of transcription.
Polycistronic DNA
Two or more genes transcribed onto the same mRNA.
Operon
regulatory regions, one promoter, and multiple genes.
basal level transcription
the gene is OFF but some small levels of protein are still transcribed.
Lac Operon Bacteria
Glucose present and no Lactose has only basal level transcription of lactase.
Lactose present, Repressor binds effector and come off. More transcription.
Glucose absent and Lactose present has repressor off and activator bound for high transcription.
DNA binding protein motif
Zinc finger, Leucine zipper, Helix-turn-helix, & Helix-loop-helix
Regulatory proteins
bind to their specific DNA sequence as dimers.
Post transcription regulation
RNA double strands: use a dicer to cut Prucursor RNA ( sing stranded RNA that has looped on self), or RNAi (double stranded RNA). Dicer cuts to make miRNA or siRNA which can silence mRNA by creating double helix
N-terminal Amino Acids
Some amino acids degrade quicker than others. One way to control a protein already made
Signal Trasnduction
Phosphorylation cascades can lead to gene regulation in the cell through endocrine system.
LacI
Inducer. Gene for the lac repressor
LacO
Operator (where repressor binds)
LacZ
Gene for β galactosidase (protein that digests lactose)
LacY
gene for permease (which brings lactose into the cell)
Allolactose
isomer of lactose and acts as the inactivating effector for the lac repressor.
Constitutive
the genes are continually expressed
Inducible
the genes can be regulated by the repressor + operator so that we make the proteins when needed.
CRP-cAMP
CRP is the activator for lac operon and ara operon. cAMP is the effector. When bound, transcription increases greatly.
AraC
dimer that is effected by arabinose. Goes from a repressor (negative regulation) to activator (positive regulation) once bound with arabinose,
