Powerpoints & Study Guides Flashcards
DNA replication requires what enzymes?
helicase, primase, DNA polymerase, DNA ligase, topoisomerase
Small genomes have
a single replication origin
DNA is synthesized in what direction?
5’ to 3’
What is a dNTP?
a building block and energy source for a polymerase catalyzed reaction
DNA primase is
an RNA polymerase
What does primase do?
Initiates DNA replication by laying down an RNA primer
What does helicase do?
unwinds parental DNA
What does topoisomerase do?
takes the supercoils out of the parental DNA molecule
Before the fork, which enzyme is first?
topoisomerase
E. coli DNA polymerase III is
the main DNA synthesis enzyme
E. coli DNA polymerase III makes
the leading strand and Okazaki fragments
E. coli DNA polymerase has ___ activity
3’ to 5’
What is the point of 3’ to 5’ exonuclease activity?
corrects 99% of errors of polymerase
E. coli DNA polymerase I is the
DNA repair polymerase
E. coli DNA polymerase I has both
5’ to 3’ and 3’ to 5’ exonuclease activity
What is the point of 5’ to 3’ exonuclease activity?
removes RNA primers
What does DNA ligase do?
seals gap in sugar-phosphate backbone left behind by DNA polymerase I
DNA polymerase binds as a
dimer
From the origin, replication proceeds bidirectionally, yielding
two replication forks
Bacterial chromosomes have how many replication origins?
1
What’s the special DNA sequence found at the origin of replication?
A-T rich region
There are more/fewer DNA polymerases in eukaryotes than in prokaryotes
More
The DNA pol a/primase complex synthesizes
a short RNA/DNA hybrid
Telomerase is
a DNA polymerase
What does telomerase do?
carries an internal RNA template that directs the synthesis of nucleotide repeats
DNA in eukaryotic chromosomes is
wrapped around histones (H2A, H2B, H3 and H4)
Germ-line mutations affect
the entire organism
Can germ-line mutations be passed on?
Yes
Somatic mutations affect
a patch - the earlier the mutation, the larger the patch
Can somatic mutations be passed on?
No
Aneuploidy is when there are
too few or too many chromosomes
A point mutation is
a change in a single base pair
What is a transition?
It’s a type of point mutation where a pryimidine (C or T) changes to another pyrimidine or a purine (A or G) to another purine
What is a transversion?
A type of point mutation where a pyrimidine changes to a purine or vice versa
Are transitions or transversions more common?
Transitions
Mutations distort the
double helix
What can happen if there’s a mutation on the promoter?
The rate of transcription may be increased or decreased
What can happen if there’s a mutation on the regulatory element/operator site?
The gene may not be able to be properly regulated
What can happen if there’s a mutation on 5’ UTR/3’ UTR
May alter the ability of mRNA to be translated and it’s stability
What can happen if there’s a mutation on the splice recognition sequence?
May alter the ability of pre-mRNA to be properly spliced
What are the two most frequent spontaneous reactions that cause serious DNA damage?
depurination and deamination
What is depurination?
the process by which purine bases are lost because their N-glycosyl linkages to deoxyribose are spontaneously hydrolyzed
Where does deamination occur?
All bases except thymine
What is the most common type of deamination?
cytosine to uracil
What is deamination?
The loss of an amine group - is replaced with a doubly bonded oxygen
What is a tautomeric shift?
A nucleotide changes from a keto to an enol form
Which form is more common? Keto or enol?
Keto
What is the result of a tautomeric shift?
Fucked up base pairing; tautomeric shifts during DNA replication can cause mutations
What does TNRE stand for?
trinucleotide repeat expansion
What happens in people with TNRE disorders?
The length of a TNR increases above the critical size
What is a potential cause of TNRE?
DNA polymerase slipping on the template
What is one example of a human genetic disease caused by TNRE?
Huntington’s disease
What effect does UV light have on DNA?
promotes pyrimidine dimer formation (thymine dimers)
What effect do E-rays have on DNA?
can cause base deletions, single-stranded nicks in DNA, crosslinking, and chromosomal breaks
What are DNA adducts?
chemical groups that are added onto DNA as a consequence of high exposure to a mutagenic chemical
What are the two ways to repair double-stranded breaks?
Homologous recombination and non-homologous end-joining
What are the four ways to repair point alterations?
direct repair, base excision repair, nucleotide excision repair, and mismatch repair
What enzymes are involved in base excision repair?
N-glycosylase, AP endonuclease, DNA poymerase, DNA ligase
What complex tracks along DNA, searching for damage?
UvrA/UvrB complex
When UvrA/UvrB complex detects damage, what happens to the complex?
UvrA is released and UvrC binds
What does UvrC do with damaged DNA?
makes cuts on both sides of the damage
What does UvrD do?
removes the damaged DNA that UvrC just cut out
What enzyme fills in the gap left by UvrD?
DNA polymerase - DNA ligase seals the gap
What complex deals with mismatch repair?
MutS/MutL
Which protein finds a mismatch?
MutS
What does MutH do?
Binds to hemimethylated sequence and makes a cut in the nonmethylated strand. The complex binds to MutH also
What does MutU do?
Separates the DNA strands at the cleavage site
How does mismatch repair end?
DNA polymerase fills the gap left behind and DNA ligase seals it
What kind of mutation results in Xoderma pigmentosum?
Nucleotide excision repair
What are restriction enzymes?
endonucleases
Which class of restriction enzyme have a palindromic recognition sequence?
Class II
Restriction enzymes leave
blunt or sticky ends
Type I restriction enzymes are not used much as they are
complex, multi-subunit, combination restriction-and-modification enzymes, randomly cut DNA far from the recognition site
Type II restriction enzymes are commonly used to
cut DNA at defined positions close to the recognition site
Type III restriction enzymes are sometimes used to
cut outside of the recognition sequence and require two in opposite orientations within the same DNA molecule
Type IV restriction enzymes can recognize
modified (usually methylated) DNA
What does EcoRI do?
creates 4 nucleotide sticky ends with 5’ end overhangs of AATT
What color is a colony if it contains the insert from a recircularized vector?
white
What is Sanger DNA sequencing?
dideoxy method!!!
How does Sanger/dideoxy chain termination work?
the chain stops growing at a specific nucleotide that is missing a 3’ OH group
Tagged bases randomly terminate
chains at specific nucleotides, denoted by a specific label (color)
What 4 things are needed for PCR?
Template DNA, Oligonucleotide primers, deoxynucleoside triphosphates (dNTPs), and Taq polymerase
What are oligonucleotide primers?
Complementary to sequences at the ends of the DNA fragment to be amplified
What are the super vague steps of PCR?
Denaturation (double stranded DNA + heat = single strands), primer annealing (lower temperature), extension
How many copies of DNA can be made with PCR?
a shit ton. like a billion
What is gene editing?
The process by which a specific, predetermined modification is made to DNA in living cells
Gene editing using what type of double stranded break repair?
Homologous recombination
What are the three classes of programmable nucleases to make breaks?
Zinc finger nucleases, TALENs, and CRISPR
What’s the point of targeted mutagenesis through non-homologous end-joining?
Introduces an insertion or deletion mutation at the break site - can cause a frameshift
How long should a PCR primer be?
18-22 nucleotides
In nucleic acids, what are connected by covalent glycosidic bonds?
Sugars are connected to bases by glycosidic bonds
How is the sugar in RNA different than the sugar in DNA?
The sugar in DNA is missing the 2’ OH group
How are the two strands of a DNA molecule held together?
hydrogen bonds between the bases
What are the purines?
Adenine and guanine
What are the pyrimidines?
cytosine, thymine, and uracil
Why do DNA binding proteins usually recognize the bases through the major groove?
All four bases can be distinguished in the major groove
The backbone of a DNA molecule is composed of alternating
sugar and phosphate groups
Which nucleic acid contains many modified bases?
tRNA
Why do the ends of all your chromosomes have the same sequence?
They’re synthesized by telomerase
What type of enzyme is DNA primase?
RNA polymerase
Which DNA polymerase does most of the DNA synthesis?
DNA polymerase III
What is trisomy?
An organism has three copies of a specific chromosome
What is triploidy?
An organism has three complete sets of chromosomes
What are three things that are the same between transcription and DNA replicatin?
RNA and DNA are synthesized in the 5’ to 3’ direction, both require the DNA to be unwound, and both only begin at specific sites on the DNA
For transcription, which strand is the template strand?
The strand that is transcribed
The sense strand has the same sequence as the
transcript, except there is a U where there was a T before
Is a primer needed for transcription?
No, RNA polymerase doesn’t need it
Does transcription require helicase or topoisomerase?
No because only a small part of the DNA needs to be unwound at a time
Different genes are transcribed at the same/different rate
different
What are regulatory sequences (DNA)
site where regulatory proteins bind, influences the rate of transcription
What is the promoter?
a site on the DNA for RNA polymerase to bind, which signals the beginning on transcription
What is the ribosome-binding site on mRNA?
the site where the ribosome binds; translation begins near this site
What is the start codon?
specifies the first amino acid in a polypeptide sequence
What does it mean to be polycistronic?
When an mRNA encodes 2+ polypeptides
E. coli promoter region includes
-35 sequence to +1 transcriptional start site
What does the sigma factor do?
Recognizes a promoter as it slides along the DNA with RNA polymerase
The Rho protein is
a helicase
What is a constituitive gene?
The gene is expressed at a pretty constant level all the time
Transcriptional regulation is the most common way to
regulate gene expression
Repressors bind to ___ and ___ transcription
DNA, inhibit
Repressors are an example of ___ control
negative
Activators bind to ___ and ___ transcription
DNA, increase
Activators are an example of ___ control
Positive
Small effector molecules bind to
regulatory proteins (repressors and activators)
What are three types of small effector molecules?
Inducers, co-repressors, and inhibitors
Corepressors activate
repressors - make them repress MORE
Inhibitors inhibit
activators - makes an activator not work, so no transcript is made
What’s an operon?
A regulatory unit consisting of a few structural genes under the control of one promoter
What are the conditions for efficient utilization of lactose?
Lactose is present and glucose is absent
What are the three structural genes of the lac operon?
lacZ, lac Y, and lacA
What does lacZ encode?
B-galactosidase
What does lacY encode?
lactose permease
What does lacA encode?
transacetylase
Four DNA elements of the lac operon
Promoter, operator, CAP site, and terminator
What does B-galactosidase do?
cleaves lactose and lactose analogues, converts lactose to allolactose
What does lactose permease do?
it’s a membrane protein required for transport of lactose and analogues into the cell
What is the lacI gene?
it encodes the lac repressor and has it’s own promoter (i promoter)
The lac operon is ___ and ___ regulated
negatively, positively
The lac operon is inducible by
lactose, CAP-cAMP
What happens to cAMP levels when glucose is around?
They decrease
Lactose is present, glucose is not
cAMP is high
Repressor is off
Activator is on
Yes transcription
No Lactose, no glucose
cAMP is high
Repressor is on
Activator is on
No transcription
Lactose is present, glucose is present
cAMP is low
Repressor is off
Activator is off
No transcription
Glucose is present, no lactose
cAMP is low
Repressor is on
Activator is off
No transcription
The lac operon is only expressed when
the repressor is off and the activator is on
A loop in the DNA brings what sites closer together?
Operator sites
Five structural genes of the trp operon
trpE, trpD, trpC, trpB, and trpA
Regulatory genes of the trp operon
trpR (repressor) and trpL (encodes the leader peptide)
High typtophan levels cause
attenuation
The promoter for eukaryotic transcription is
the TATA box and variable sets of additional DNA elements
What proteins are required for initiation in eukaryotic transcription?
basal transcription factors (lots)
RNA processing that occurs in prokaryotic transcription
none
RNA processing that occurs in eukaryotic transcription
capping, polyadenylation, and splicing
RNA polymerase I transcribes
45S pre-rRNA in the nucleolus
RNA polymerase II transcribes
mRNA, snRNA, miRNA in the nucleus
RNA polymerase III transcribes
tRNA, 5S rRNA in the nucleus
Prokaryotic Pribnow box
-10 TATAAT
TFIID is a complex of
proteins, includes TATA binding proteins (TBP) and TAFs
TFIID binds to
TATA box
What binds after TFIID binds to TATA box (second step)?
TFIIB binds
What does TFIIB do?
Acts as a bridge to bind RNA polymerase II and TFIIF
What forms the preinitiation/closed complex?
TFIIE and TFIIH binding to RNA polymerase II
TFIIH forms
an open complex, phosphorylates CTD domain
True or false: enhancers are close to the basal promoter
false
Activators bind to
enhacers
TATA binding protein binds to the TATA box and causes these two changes in DNA structure
two bends and a region of unwinding between them
Two main jobs of TFIIH
unwinds DNA helix, Cdk7 phosphorylates the carboxy terminal domain of RNA polymerase II
Where can enhancers not be located?
In the promoter proximal region
Transcription activation domains work by recruiting
RNA polymerase II to basal promoter through interactions with general transcription factors
Co-activators are
non-DNA binding proteins that serve as bridges between activators and the basal transcriptional machinery
What does the Id protein do?
produced during early stages of development, prevents MyoD from promoting muscle differentiation too soon
Without Id, what happens to MyoD?
Forms heterodimers with bHLH factors, allowing the heterodimer to bind to enhancer in the promoters of muscle-specific genes
Changes in chromatin structure affect
transcription
4 types of changes that occur in chromatin structure
DNA methylation, nucleosome spacing changes, nucleosome replacement, and histone modification
Methylation can inhibit
the binding of activators - methyl group blocks the binding of an activator to an enhancer
Where does methylation occur?
CpG island
Nucleosome spacing changes affect
the relative spacing and/or positions and/or number of nucleosomes
How does histone acetylation affect the strength of DNA binding?
Weakens it - DNA is less tightly bound to acetylated histones
The affects of methylation of histones on transcription depend on
which histone is methylated and where
Are histone modifications reversible?
Yes
What’s the role of the mediator?
Brings activator proteins that bind to more distant regulatory sequences of the DNA in contact with the initiation complex - binding of the mediator triggers phosphorylation of the CTD
What are the four DNA-binding domains?
Helix-turn-helix, helix-loop helix, zinc finger, and leucine zipper
In eukaryotes, what event triggers the conversion of the “closed complex” to an “open complex” and the start of transcription?
Phosphorylation of the CTD of RNA polymerase II
What type of histone modification can inhibit transcription?
Methylation and deacetylation
Name one example of a protein that binds DNA and bends it
restriction enzymes, TBP
In prokaryotic RNA, a hairpin structure followed by a strong of Us causes what?
termination of transcription
What protein is involved in termination in prokaryotes?
Rho
Which DNA strand is transcribed?
Template strand
What enzyme modifies histones?
Histone acetylase
If the template strand is 5’ GCCGCC 3’ what is the mRNA?
5’ GGCGGC 3’
What is the DNA region that contains binding sites for specific transcription factors?
promoter proximal region
3 different splicing mechanisms
Group I intron splicing, Group II intron splicing, spliceosome
Ribosomal RNAs are cleaved out of
precursor RNA
1st step of mRNA processing
5’ cap
What is added to the 5’ end of pre-mRNA?
non-templated G residue
1st step of the formation of the 5’ cap
a phosphatase removes a phosphate from 5’ end
2nd step of the formation of the 5’ cap
guanylyl transferase adds GMP as a 5’-5’ triphosphate linkage
3rd step of the formation of the 5’ cap
G is modified by methyl transferases - adds methyl groups
2nd step in mRNA processing
3’ polyadenylation
What are the two polyadenylation factors?
CPSF and CStF
What does the 5’ cap do?
protects the 5’ end from 5’ to 3’ nucleases
What does the poly A tail do?
protects the 3’ end from 3’ to 5’ nucleases
3rd step in mRNA processing
splicing
What occurs in splicing?
precursor RNA is made into a mature RNA and the intron is released
