Final Flashcards
Three properties of DNA
- Accurate replication
- Have info with instructions to encode for proteins
- Be able to change (rarely) so that it results in hereditary changes/mutations
What is purines made of
Adenine and Guanine
(Pure as gold)
What is Pyrimidines made of
Cytosine and Thymine
(C(U)T) the py (pie)
Phosphate of DNA binds to what?
5’ position of CH2 deoxyribose sugar
Where is the OH located on the deoxyribose sugar?
3’ position
Base pairing
A+G = T+C
A=T; G=C
Base pairing practice
If G+C of a double-stranded DNA is 48%, what is the percentage of ‘A’ in this DNA molecule
Since G+C= 48% we can find A+T
A+T= 100% - 48% = 52%
Since we know A=T we divide 52% by 2 to find A and T individually
52%/2 = 26%
Therefore A is 26%
Complementary base pairs (Chargaff’s Rule)
A-T and G-C
the sugar of DNA binds to what?
3’ end
the phosphate of DNA binds to what?
5’ end
How is DNA replicated?
Semi-conservative replication
What is semi-conservative replication?
when parental DNA strands unwind and combine for synthesis of new complementary strand
What produces energy to help DNA synthesis?
cleaving of pyrophosphate
what makes up dNTP (Deoxyribonucleoside triphosphate)?
3 phosphates, sugar group, one nucleotide
What is the first step of DNA replication?
DNA gyros relaxes DNA helices and rejoins strands
(makes sure DNA is not coiled)
What is the second step of DNA replication?
Helices distrupts H bonds thus unwinding helices
(ssB= ssDNA binding proteins that stabilizes unwound DNA)
What is the third step of DNA replication?
RNA polymerase synthesizes primers. Primers synthesize (in 5-3) RNA primers using DNA as template.
(Creates binding site for DNA Pol III)
What is the fourth step of DNA replication?
DNA Polymerase III catalyzes synthesis of DNA (starts at 3’ end of primer)
What is the fifth step of DNA replication
DNA polymerase I removes RNA primers and fills in gaps
What is the sixth step of DNA replication?
DNA ligase connects adjacent DNA fragments
why is DNA replication semi-discontinuous?
The lagging strand needs to be synthesized in small sections.
(DNA Pol III bind to RNA primers and synthesizes Okazaki strands. DNA ligase needs to connect adjacent DNA fragments)
what does the OH on the sugar do to the triphosphate?
a nucleophilic attack on an incoming nucleotide so that it can bind to it
what are PCRs? (Polymerase chain reaction)
a method of amplifying DNA
(ex. forensics)
What is the first component for PCRs?
Isolated genomic DNA (like from hair) to act as template DNA
What is the second component for PCRs?
Multiple copies of DNA primers.
- approx 20 nucleotides long WITH FREE OH GROUP
- reverse and forward primers always written in 5-3
what is the third component for PCRs?
dNTPS (dATP, dGTP, dCTP, dTTP)
- provides single bases (A, G, C, T)
What is the fourth component for PCRs
Taq polymerase (special DNA polymerase
- acts as DNA POL III
- Synthesis in 5-3 direction
- active at high temps over multiple cycles of heating and cooling
PCR step one
Denaturation of DNA via heating
PCR step two
Annealing of primers onto appropriate locations on template via cooling
PCR step three
Tax polymerase binds to primers for synthesis of DNA via heating (adds to 3’ end)
what determines annealing temperature?
depends on melting point (mp) of primers. temp should be lower than mp
What determines melting point of PCRs?
- melting point is when 50% of primers are bound and 50% of primers aren’t bound
- more GC = higher temp
- longer dna = higher temp
what is the difference between DNA Pol I and DNA Pol III?
DNA Pol I removes RNA primers and fills in gaps
DNA Pol III synthesizes DNA
designing forward and reverse primers
- Forward primers is always the same as the top strand (from 5’ end)
- the reverse primers is the compliment of the top strands (from 3’ end and backwards)
what is gel electrophoresis
method used to separate DNA (or RNA and proteins) molecules based on their sizes by using electrical field to move molecules through gel matrix
what is agarose primers?
used to make ‘gel’ to separate DNA
what DNA molecules will travel quickly through the gel matrix?
small DNA molecules because they have lower molecular weight
what DNA molecules will travel slower through the gel matrix?
larger DNA molecules bc it has higher molecular weight
why do we stain DNA in gel electrophoresis?
- to see DNA under UV light bc DNA is colourless
what are molecular ladders?
DNA pieces with known sizes to help determine size of DNA samples
What are SNPs?
single base pair difference between DNA sequences (can be transition or transvehrsion)
what is transition?
replaces pyrimidine with pyrimidine
replaced purine with purine
what is transversion
replaces purine with a pyrimidine or vice versa
what is spontaneous DNA replication error?
- a cause of SNPs
- mismatching
- can be caused by tautomerization
what is tautomerization?
changes in position of a base’s atoms and bonds between atoms
how are mismatched errors repaired?
- by the DNA Pol III or I
- remove mis-paired bases by working backwards (3-5) (3-5 exonuclease activity)
- if mistake is still there, its usually repaired by other DNA repair mechanism
what is “other chemical changes to nucleotide?”
- a cause of SNPs
- can be caused by depurination or deamination
what is depurination?
- loss of purine base
- can cause polymerase to stop or add random base
(phosphate and sugar backbone still intact)
what is deamination?
removal of amine groups (cytosine, adenine, and guanine)
what are insertion-deletions (Indels)?
- insertion or deletion of bases
(Deletion of bases results in no frameshift) - can result in non-repetitive indels (point mutations) or STRs (short tandem repeats of 2-9 nucleotide)
what is point mutation?
single base pair being added, deleted, or changed (SNPs)
More info about STRs?
- also called micro satellites- repeated region could be exon, intron, regulatory regions, nonfunctional DNA sequences
- high mutation rates
what is slippage?
when a CAG repeat folds in and causes right side (of CAG) to be repeated since it fell backwards
what os the sufferance between dideoxynucleoside and deoxynuclooside?
Dideoxy has H on the 3’ position while Deoxy has OH on 3’ position
How is PCR different from Sanger Sequencing?
- PCR results in exponential amplification (sequencing does not)
- one primer is needed in Sanger
(PCR needs 2) - Sanger needs ddNTPS and dNTPs
In Sanger sequencing, which base is always causes the termination of synthesis?
In T tube: T
In C tube: C
In G tube: G
In A tube: A
how are RNA and DNA different (structurally?)
- RNA sugar has OH on the 2’ carbon while DNA has H on the 2’
- RNA has U (no CH3 group) and DNA has T
(thymine is more stable)
what direction does RNA synthesize in?
5’ to 3’ direction
what direction does the RNA polymerase reads the template strand? (in RNA transcription elongation)
3’-5’ direction
Newly synthesized RNA is made in what direction?
5’-3’ direction
What does the RNA polymerase do
- unwinds about 17bp of DNA
- catalyzes RNA synthesis
what does upstream mean
The region that is closer to the 5’
what does downstream mean
the region towards the 3’ end
what happens in RNA transcription?
- The DNA has regulatory sequences (enhancer/silencer, promoter), UTRs, exons, and introns
- Splicing splices off all regulatory proteins leaving only start/stop codons, exons, introns, UTRs and terminator. (Turns into pre-mRNA)
What happens during RNA translation?
- introns gets splices out leaving only exons, UTRs, and terminator
- then gets turned into protein
How does the RNA know where the introns are to splice them off?
- the spliceosome recognizes specific nucleotide sequence at exon and intron junctions
- Introns begins with GU and end with AG
at what position/site does transcription start?
+1 site
what is the non-template strand?
- sense or coding strand
- 5’-3’
what is the template strand?
- antisense or noncoding strand
- 3’-5’
Where is the TATA box and what does it do?
- positions -25 to -35
- positions the RNA polymerase for initiation of transcription
what is quantitative PCR?
- method of PCR allowing us to quantify the amount of DNA in sample (by using fluorescent dye)
- used for COVID19 testing
what is CT?
amount of cycles needed to pass the threshold
what does low CT value mean?
More DNA is present bc it takes less cycles
what does high CT value mean?
Less DNA is present bc it takes more cycles
what forms between amino acids?
peptide bonds
(genetic code) what does it mean when codes are degenerate?
- multiple codons encode the same amino acid
- helps buffer against transition SNPs
N terminal on a protein does what?
- amino group
- synthesize first
C terminal of a protein does what?
- carboxyl group
- synthesize last
what are tRNAs?
- act as adaptors (or links) between mRNA and amino acids
- has anticodon loop (3’-5’)
- animo acid attached to 3’ end
what does aminoacyl-tRNA synthetase do?
its an enzyme that attaches amino acids to tRNAs
what does ribosomes do?
- bring together the charged tRNA and mRNA
- move along mRNA in 5-3
what does the peptide-transferase centre do?
catalyze the formation of peptide bonds between amino acids
what is the shine-dalgarno sequence and kozak sequence and how are they different?
- both are sequences in mRNA that the rRNA recognizes (helps position the ribosome)
- kozak sequence is found in eukaryotes
- shine-dalgarno sequence is found in prokaryotes
what are the 4 outcomes of SNPs in exons?
- silent mutations
- missense mutation (conservative)
- missense mutation (non-conservative)
- nonsense mutation
what is silent mutation?
- synonymous (same)
- encodes same amino acid
what is missense mutation (conservative)
encodes a chemically similar amino acid
ex. AAG (Lys) –> AGG (Arg)
what is missense mutation (non-conservative)?
encodes a chemically different amino acid
ex. AAG (Lys) –> ACG (The)
what is nonsense mutation?
encodes a stop codon
how can insertions and deletions in exons impact amino acids?
can cause frame shift mutation
where is the shine-dalgarno and kozak sequence usually placed?
after +1 poisition
what is a Western Blot?
- it is used to detect particular proteins in a mixture of proteins
- can determine size of proteins and lvl of expression
what does a probe (in Western Blot) do?
they are antibodies that bind to protein of interest
- secondary antibody binds to primary antibody and has fluorescent body
- primary antibody binds to protein of interest
in terms of western blot, “expansion” means
more bp = bigger = towards the top
in terms of western blot, “increased expression” means
bigger protein spot (bigger circle) bc there is more protein for antibody to bind to
where is the promoter positioned?
upstream and before position 1+
RNA polymerase in prokaryotes (E.coli)
- made of 5 subunit core and a subunit of sigma factor
- sigma factor leaves after initiation
what is the role of the sigma factor?
to position enzyme correctly at promoters
what are the two transcription termination in E. coli?
- factor-independent termination
- Rho-dependent termination
what is factor-independent termination?
A G-C rich DNA followed w/A-rich stretch causing RNA polymerase to release from DNA template
what is Rho-dependent termination?
Rho-factor (protein) recognizing c-rich sequence and releases RNA polymerase from DNA template
Regulation of gene expression in bacteria
- needs to breakdown sugar from enviro (uses sugar as carbon source)
- conserve energy by making enzymes required for import + metabolism when sugar is present
what is the role of the activator protein?
brings RNA polymerase closer to promotor for transcription
what is the role of the repressor protein
it blocks RNA polymerase from binding to promoter or blocks it from moving along DNA
what does removing the activator protein do?
causes no transcription or basal/low transcription
what is the role of allosteric effectors?
they bind to allosteric sites on regulatory proteins and changes the conformation of those proteins.
- activator effectors allow protein to bind to promoter therefore have transcription
- repressor effectors changes conformation of repressor protein making it unable to bind to operator (allowing transcription)
What is permease (Y)?
- type of lactose operon, structural gene (protein coding gene)
- transports lactose into the cell
what is Beta-galactosidase (Z)?
- type of lactose operon, structural gene (protein coding gene)
- modifies lactose into allolactose + cleaves lactose
what is an “I” gene?
encodes a repressor
What is trans-acting regulator elements?
affects gene expression of distant genes (can be on other DNA)
what is cis-acting regulator elements?
affects gene expression of nearby genes (on same DNA)
ex. promotor/operator both only affect things its physically attached to
what does negative regulation require?
always involves a repressor
what is allolactose?
an allosteric effector causing repressor to be unable to bin to operator
what is activator effector?
a type of allosteric effector that allows regulatory protein too be able to bind to promoter
what is repressor effector?
type of allosteric effector that binds to the repressor and makes it unable to bind to the operon
In negative regulation, what happens if there is NO lactose?
lac operon = off = no expression bc a repressor is encoded
in negative regulator, what happens if there IS lactose present?
lac operon = on = transcription bc allolactose is made (repressor effector)
what is a constitutive mutation?
gene is expressed (no control over its expression)
operon is an example
what is Oc?
repressor cannot bind to operator.
what is an inducer?
it allows stuff to be turned on
Oc is epistatic (masks effect) of what?
Is
if glucose is present, what is the expression from the lac operon?
minimal expression
if glucose is NOT present, what is the expression from the lac operon?
enhanced expression (bc cell needs to work harder to break down lactose)
What is cAMP?
An allosteric effector for CAP
What is CAP?
(catabolite activator protein)
- cap-cAMP complex enhances transcription
define attenuation
level of regulation that occurs after transcription has been initiated
- high levels of Trp = low mRNA production
(In Trp operon)
In tryptophan operon level 1 regulation, what happens if there is tryptophan?
Trp will bind to TrpR repressor to bind to TrpO (opressor) and inhibit transcription
In tryptophan operon level 1 regulation, what happens if there is NO tryptophan?
no Trp will bind to TrpR therefore transcription will happen
In tryptophan operon level 2 attenuation, what happens if there is A LOT of trp?
there is a 3+4 pairing seen in the loop. It signals for termination (also has a string of U after region 4)
(transcription and translation are happening at the same time bc it is prokaryote)
In tryptophan operon level 2 attenuation, what happens if there is LOW trp?
regions 2 +3 pair continuing transcription
ribosome stalls at TRP codons in the leader peptide bc leader peptide is incomplete
what does general transcription factors in transcription initiation in eukaryotes do?
binds to specific sequences (ex. TATA box, +1 site, etc) in the promoter to help RNA Pol II bind to correct site
what does the preinitiation complex (PIC) in transcription initiation in eukaryotes do?
- it is a complex made of RNA Pol II and general transcriptional factors
- when RNA Pol II bind to general transcription factor, the C-terminal domain (CTD) of the pol is phosphorylated to facilitate processing of 5’ end of mRNA
- caps the m7G (at 5’ to decrease degradation)
why do we cap the m7G?
- protects mRNA from decay from exonuclease
- addition of methylated G signals start of transcription
- assists in splicing and translation
what is polyadenylation?
conserved sequences in 3’UTR signalling for termination of transcription with addition of poly-A-tail
what does the poly-a-tail do?
protects from decay and assists in translation
how do you get a variety of proteins?
Variety of proteins can be achieved by splicing the exons and introns out in different ways
what is the polyadenylation signal?
- part of the 3’UTR
- processes sequence for poly-A-tail (tells transcription to stop)
what is the landing site for ribosomes in mature eukaryotic mRNA?
5’-UTR
regulatory protein factors like transcription factors bind to what part?
DNA
regulatory protein factors like co-regulators bind to what part?
they bind to general transcriptional factors and NOT directly on DNA
what is the core promoter?
- in the +1 region where the general transcription factor (GTC) binds to to start transcription
since the enhancer is more upstream than the promoter, how is it able to influence the core promoter?
it bends so that it is close to the co-regulator
what are the enzymes involved in the import and metabolism of galactose in yeast?
Gal 2, 1, 7, 10
what happens if there is no galactose?
enzyme coding genes are not expressed
what happens if there is galactose
enzyme coding genes are expressed
where does GAL4 bind to?
the UAS (enhancer sequence) to activate transcription
which GAL proteins are always expressed?
GAL 3 and GAL 80
what represses GAL 4?
GAL 80
What are regulatory proteins in the yeast GAL system?
GAL 4, 3, 80
(regulate transcription of enzyme coding genes)
what goes under conformational change in presence of galactose?
GAL 3
what is a chromatin?
complex of DNA + protein that make up eukaryotic chromosomes
what is a nucleosome
DNA is packed around histone proteins (each nucleosome has 8 histone proteins)
what is the charge of histones? and what’s the function of its folded core and flexible tails?
- positive
- folded core controls how compact histones are
- flexible tails interact with adjacent nucleosomes and other proteins
what is heterochromatin
more compact
what is euchromatin
less compact (transcriptionally active)
what is constitutive heterochromatin
DNA that is always compact
(in regions that arent transcribed like telomeres and centromeres)
facultative heterochromatin
varies between being compact (heterochromatin) and open (euchromatin)
Chromatin modification: Histone modification
- primarily affects tails
- example: Histone acetylation
what is histone acetylation?
addition of acetyl group to lysine amino sic
what are the effects of histone acetylation?
- reduces chromatin compaction (loosening interaction between histones and DNA)
- creates binding site for protein domain )transcriptional activators) called bromodomain
what is bromodomain?
found in many transcriptional activators and it turns on genes (reads it)
(coregulator bc it binds to histone and not DNA)
what is the protein code hypothesis?
different combos of histone modification specify different transcription outcomes
what is HAT (histone acetyltransferase)?
writes genes
what is HDAC (histone deacetylase)?
gene eraser
Chromatin modification: DNA Modification Histones
methyl group gets added to cytosine (making every C be methylated)
what is unmethylated CpG
- in expressed genes
- acts as transcription controller
- associated with open chromatin and active transcription
methylated CpG
- in unexpressed genes
- acts as transcription controller
- is not recognized by RNA
- associated with closed chromatin and repressed transcription
what does methylation do?
- prevents transcription factor from binding
- creates binding sites for HDACs (histone deacetylase)
If a parent strand has a methylated C (cytosine), would the new strand have the methylated C or no?
The new strand will have the methylated C. The new strand will see its parent strand having it so it will change its C to have it
what does imprinting mean?
gene is silenced through methylation (so other parent trait will be expressed)
what are the steps when cloning insulin?
- obtain an expression plasmid (vector)
- obtain human insulin gene (cDNA) (cut with restriction enzymes)
- insert cDNA into expression plasmic (perform restriction map to confirm)
- introduce recombinant plasmid to bacteria (bacteria will produce insulin protein which will be extracted and purified)
what are restriction enzymes?
they are endonuclease that cleave phosphodiester bonds between nucleotides at specific DNA
why is ligating with sticky ends more compatible?
pieces of DNA cut with same restriction enzyme can be ligated together
(newly ligated DNA recreates original restriction site)
why is ligating with blunt ends less compatible?
any piece of DNA can be ligated together but the efficiency is lower
single enzyme cloning
- insertion can happen in either orientation
double enzyme cloning
- insertion only occurs in one orientation
what are some essential features for cloning?
- origin or replication (ori)
- antibiotic resistance gene
- Polylinker (multiple cloning sites)
what is the origin of replication (ori)
allows plasmids to replicate in bacteria host cell
what does the antibiotic resistance gene do?
allows only cells with plasmids to grow
what does the polylinker (multiple cloning site) do?
region containing many unique restriction enzyme sites
what are some optional features for cloning?
- promoter (required for expression plasmids)
(found upstream of polylinker) - (if you want an expression plasmid) 3’UTR, 5’UTR, shine-dalgarno, terminator
inherited means what?
DNA replication
what is morphogen?
- molecule whose effects are concentration-dependent (established a pattern of tissue development and body plan formation)
- determines hox gene expression
what are hox genes?
- genes responsible for regulation of animal development
- controls identity of segments and appendages
- encodes transcriptional factors
classes of toolkit genes
- Maternal effect genes (bicoid and nanos)
- gap genes
- pair-rule genes
- segment-polarity genes
- Hox genes
(2-3 and zygotic ally expressed)
Class of toolkit gene 1: Maternal effects gene
- established AP axis
- bicoid and nanos (codes transcription factors to regulate next genes) mRNA tethered to anterior and posterior
- proteins express gradient after fertilization
- if mom is mutant, all offspring is mutant
Class of toolkit gene 2: gap genes
- segmentation genes
- divide embryo into broad regions
- define regional sections
Class of toolkit gene 3: Pair-rule genes
- segmentation gene
- affects development of pairs of segments
- acts in more narrow regions than gap genes
- define individual segments
Class of toolkit gene 4: segment-polarity genes
- segmentation gene
- affects organization of segments (some segments may be facing opposite directions if there was a mutation)
- define organization of individual segments
Class of toolkit gene 5: hox genes
- determine identity of structure or segment
- 8 hox genes in flies
- order of genes in chromosome corresponds to order of body regions
- regulate expression of networks of other genes
IMPORTANT
- sequence specific DNA binding proteins
- bind to cis-acting regulatory elements of other genes to regulate or repress
what are gap genes activated by?
- maternal-effect proteins
- bicoid protein (transcription factor) localized in anterior
- ## bicoid only binds when there is high concentration
what is the hunchback gene?
- it is a gap gene that is expressed by high bicoid lvls
toolkit gene expression is regulated by what?
- transcription factor binding to cis-acting regulatory genes
- earlier transcription factors regulate genes expressed later
- results in genes being expressed at discrete regions
expression of pair rule gene: even skipped (eve)
- cis acting regulatory elements (enhancers) controlling eve gene bound by precise combos of transcription factors
how are genes expressed in precise patterns at different times?
- most toolkit genes function at more than one time and space
- function are context dependent
( some expression can repress/promote other genes)
what is CRISPR
a gene editing tool
Step 1 and 2 of CRISPR
acquire spacers (foreign or viral DNA) and incorporate it into genomic CRISPR region
Step 3 and 4 of CRISPR
CRISPR region gets transcribed to generate guide RNA (gRNA)
gRNA will assemble with Cas9 gene to guide it to cleave (introduce double stranded break) the invading DNA with precision
where must the target sequence be in CRISPR for the Cas9 to cleave it?
3 nucleotides upstream of PAM (5’NGG)
What is Cas9?
- inherited enzyme that makes cut in invading enzymes so that it wont infect
what’s the first way to fix a doublestranded break?
homologous recombination (HR)
- Introduces donor DNA that will be used as template to repair double stranded break
what is the second (and less efficient) way to fix a double stranded break?
Nonhomologous ending joining (NHEJ)
- Introduces deletions/mutations that can inactivate genes
