Midterm #2 Flashcards
what is DNAs function
- it carries the genetic instructions required for the expression of traits in all known living organisms
- it serves as the hereditary unit of life
what is a double helix
two spiral backbones
describe what it means for DNA to be in a double helix
DNA is a twisted ladder with the rungs being the nitrogenous bases (A, C, T, G) aka the sugar phosphate backbone
what are the individual units of DNA (nucleotide)
individual units of DNA are made up of nucleotides
1 phosphate
1 sugar
1 nitrogenous base
what holds the double helix together
hydrogen bonds (IMFs) hold the two strands together
what are the complimentary base pairs
nucleotides follow complimentary base pairing rules:
- adenine pairs with thymine
- guanine pairs with cytosine
nucleic acid
large biomolecule composed of many nucleotides (DNA and RNA)
what is a nucleotide
can refer either to deoxyribonucleotide –> monomer of DNA
ribonucleotide –> monomer of RNA
draw a nucleotide
see first page of notes
draw cytosine
see first page of notes
draw thymine
see first page of notes
draw uracil
see first page of notes
draw adenine
see first page of notes
draw guanine
see first page of notes
which nitrogenous bases are purines
adenine and guanine
what nitrogenous bases are pyrimidines
cytosine, thymine, and uracil
what are the bonds in a nucleotide
- phosphodiester bonds
- glycosidic bond
how do the nucleosides differ from ribonucleoside and deoxyribonucleoside
ribonucleoside
- found in RNA
- name (ex. cytosine)
- possess two OHs on the bottom of the ring (pg. 2)
deoxyribonucleoside
- found in DNA
- name (ex. deoxycytosine)
- possess one OH on the bottom of the ring
when a new strand of DNA or RNA is being synthesized, which side is it added to?
synthesized from 5’ to 3’
5’ has the phosphate 3’ has the sugar
(see pg. 2)
function of DNA polymerase
hydrolyzes dNTPs between alpha and beta phosphate
DNA polymerases are enzymes that create DNA molecules by assembling nucleotides
how do two nucleotides link
the hydrolysis of a high energy bond provides the energy necessary to link the two nucleotides together
what does ATP break down to
ATP often breaks down to ADP and an inorganic phosphate
during DNA synthesis, what is released as a result of hydrolysis
pyrophosphate is released
when one phosphodiester bond is created, what happens
another phosphodiester bond is created (pg. 3)
describe the base pairing of DNA
DNA has complimentary and antiparallel base pairing
what are watson crick bonds
adenine (A) forms a base pair with thymine (T) using two hydrogen bonds, and guanine (G) forms a base pair with cytosine (C) using three hydrogen bonds
draw the hydrogen bond acceptors and donors on the watson crick bonds
see pg. 3
what handedness does DNA have
right handed (bottom of the staircase, left hand on the wall, going up) –> imagine a screw
describe the major and minor grooves
- grooves occur from the way the two phosphate sugar backbones curve
- angle of the gycosidic bonds are what create major vs. minor grooves
- smaller angles make the minor grooves
length of major and minor grooves
~10 nucleotides
the length of one turn is 3.4 nm (34 angstroms)
where does the DNA binding proton bind to
the major groove of DNA via hydrogens
- this is because the major groove is chemically rich
what is the significance of the major groove
allows unique DNA sequences that allow for specificity for DNA binding proteins (ex. turning on/off genes)
what is tautomerization
unstable isomer caused by the shift of a hydrogen
- results in alternative base pairing may cause a change in the sequence (mutation)
what tautomer does thymine bond to
enol tautomer of guanine
what tautomer does guanine bond to
enol tautomer of thymine
what tautomer does adenine bond to
imino tautomer of cytosine
what tautomer does cystosine bond to
imino tautomer of adenine
draw where the hydrogen goes from the amino to the imino form
pg. 7
draw where the hydrogen goes from the keto to the enol form
pg. 7
what is the tautomerization in the TEMPLATE
pg. 7
what is the tautomerization in the SUBSTRATE
pg. 7
define gene
the sequence of DNA that codes for a trait
nucleotide
the individual molecules that make up the DNA (A, T, C, G)
double heliz
the three dimensional structure of the DNA
genome
the complete set of genetic material
chromosome
the compacted DNA structure wound around proteins (histones)
allele
the specific version of the gene
DNA
the series of nucleic acids that contains our genes
how many chromosomes do humans have
2 pairs of 23 chromosomes
- one set of chromosomes is from the mother, the other from the father
- 22 pairs of non-sex chromosomes (autosomes) and one pair of sex chromosomes
XY
phenotypic male
XX
phenotypic female
klinefelter’s syndrome
XXY chromosomes
turner’s syndrome
X chromosome
down syndrome
have a 3rd chromosome at chromosome 21 (trisomy 21)
aneuploidy
atypical number of chromosomes
draw the anatomy of homologous chromosomes
pg. 9
where are alleles located
the gene locus
what is the haploid genome
1 set of chromosomes –> ~3 billion base pairs
where is our genetic information stored
the nuclei of most of our cells in our body (except for red blood cells)
- DNA is the same in every cell (even though DNA is the same in every cell, different genes are expressed (turned on) in different cells)
what do genes make
proteins
proteins
proteins are the workhorse of the cell and they come in all varieties:
- enzymes catalyze rxns (ex. lactase breaks down lactose)
- structural proteins provide support and shape to the cell
- cell surface proteins receptors (acid binding)
why do genes typically have two different alleles (versions) of a gene
a parent contains two alleles of a gene but randomly contributes just one of those alleles to each of their offspring - offspring inherits a set of genes from mom and dad
genotype
the specific allele combination for a gene
phenotype
the outward expression of the genotype
punnet square PP
homozygous dominant
punnet square Pp
heterozygous
punnet square pp
homozygous recessive
what is the function of the dominant allele
makes working protein
what is the function of the recessive allele
makes broken protein
what do metabolic pathways do
help cells break down macromolecules, such as, proteins, nucleic acids, polysaccharides, and lipids
what do catabolic pathways achieve
important for releasing energy and heat
describe the structure of metabolic pathways
pg. 15
a precursor molecule (the substrate for the enzyme) is catalyzed by an enzyme that was encoded for by an organism. then, intermediates are formed until the product is achieved
alkaptonuria
a recessive disease hallmarked by black pigment accumulation in the tissues due to the build up of homogentisic acid. urine turns black upon exposure to air (oxidation)
how can you determine which enzyme in the metabolic pathway was broken or missing
by examining which metabolite was bulding up
how do defects in different steps of a pathway effect phenotypes
defects in different steps lead to different phenotypes
phenylketonuria
build up of phenylalanine can damage the nervous system, which leads to many symptoms
PKU and AKU are caused by blocks in steps of a pathway, what is the likely outcome of a person who is heterozygous for both genes
normal phenotype – heterozygous for each means they have a working enzyme for each, so pathway can be completed as normal.
PKU and AKU are caused by blocks in steps of a pathway, what is the likely outcome of a person who is heterozygous for both genes
some combination of both genes
how do we discover what genes are involved in a process
genetic screen
- random mutagenesis in a model organism
- search for mutations that exhibit a loss-of-function phenotype
determine gene identity of mutant
- complementation test –> only useful to determine gene identity when you have another mutant whose identity you know
- gene mapping
phototroph
wild type
- can synthesize what it needs (AAs, nitrogenous bases, or vitamins)
auxotroph
mutant
- cannot synthesize what it needs due to mutated gene in an essential metabolic enzyme; these require addition of a particular nutrient
why do we do a genetic screen for auxotroph mutants
to uncover all of the genes / enzymes for all of the steps in the pathway
conditional mutants
under one condition (the permissive condition) they exhibit wild type phenotype (grow / survive) and under another condition (restrictive condition) the show their mutant phenotype (die in this case)
- these allow you to have control on whether the mutant or wild type is shown
describe the restrictive condition for the genetic screening of conditional mutants
contains minimal media
- requires the organism to synthesize metabolites
- wild type phototrophs show growth
- mutant auxotrophs show no growth
what is minimal media composed of
Media that contains the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow “wild type” microorganisms.
describe the permissive condition for the genetic screening of conditional mutants
- contains all the metabolites
- does not require the organism to synthesize metabolites
- wild type phototrophs show growth
- mutant auxotrophs show growth
a mutant of yeast unable to make guanine should grow on what media
pg 18
minimal + nitrogenous base
what does no growth indicate
that the mutant carries mutation in the synthetic pathways for more than one amino acids
complementation test
method to determine whether two mutants with the same phenotype are alleles of the same or different genes
draw the difference between alleles with mutants in different genes vs. alleles with mutants in the same genes
pg. 21
what is it called when two mutants are in different genes
we say they compliment one another because they each can provide the wild type
fail to complement
groups that fail to complement one another because they have mutations in the same gene
draw complementing strands
pg 21
why and when must you test for dominance
test for dominance before complementation test because complementation tests are only useful for mutants that are recessive
- a test for dominance is performed by crossing the mutant with the wild type and checking first that the wild type will rescue the mutant
- if something is recessive, you will see growth on minimal media
draw recessive auxotroph in the test for dominance
pg. 22
do practice problems on pg. 22
do practice problem on pg. 23
do practice problems on pg. 24
draw the anatomy of a gene
pg. 25
what are the components of a gene
pg. 25
- control elements
- promoter
- +1 site
- 5’ UTR
- coding region
3’ UTR
describe transcription on a coding strand
pg. 25
transcription creates a replica
- transcription haults where there is a transcription termination signal (typically a long row of As)
how does space effect the coding region
pg. 25
- the extra space helps preserve the coding region
coding region
pg. 25
stretch of DNA that contains the coding sequence that will encode the protein (in eukaryotes, this includes both exons and introns, though introns will be removed)
coding strand
pg. 25
strand of DNA that contains the coding sequence of the gene of interest
UTR
pg. 25
untranslated region
- at the beginning of the 5’ UTR, we have the +1 site
- 5’ UTAR is important in facilitating contact with the ribosome
+1 site
pg. 25
represents the first nucleotide that is translated
promoter
pg. 25
region of DNA upstream of transcription start site that is recognized by RNA polymerase
draw the promoter on a region of DNA
pg. 25
control elements
pg. 25
regions of DNA that increase or decrease the rate of transcription
describe the mechanism of trancription
NN pg. 1
when transcirption is hindered, RNA polymerase can associate with the promoter by binding to another factor called the sigma factor
once RNA makes contact, transcription is initiated
the two strands of the DNA separate to make a transcription bubble
RNA pol transrcibes the DNA by reading the template from 3’ to 5’ which allows RNA pol to make an exact replica of the coding strand (replacing Ts with Us)
RNA transcribes the new transcript from 5’ to 3’
RNA pol runs into the termination site and then released from the DNA
sigma factor
kind of transcription initiation factor that helps RNA polymerase recognize the promoter
- releases once RNA polymerase makes contact and initiates transcription
draw the interaction between the prokaryotic promoter and its RNA polymerase
NN pg. 1
holoenzyme
made of an apoenzyme (RNA pol) and its cofactor (sigma factor)
apoenzyme
the inactive form of the enzyme and requires a cofactor to be active
what is the role of the sigma factor
allows RNA pol to make contact with the promoter. it then releases after RNA pol initiates transcription
what sites does RNA pol make contact with
-35 and -10 sites
what is the purpose of the space between the -35 and -10 sites
the distance of +/- 17 nucleotides of any variety provides optimal space needed for functioning
concensus sequence
sequence of DNA recognized by prokaryotic RNA polymerases
- these are the optimal sequences
- the closer the -10 and -35 sequence match the consensus sequence, the tighter the RNA polymerase binds and the more transcript that is made for that gene.
what effect do changes to DNA binding site have
any changes to DNA binding site (-10 and -35 sites) weakens the sigma factors association which weakens the RNA pol holoenzyme association. This means the the RNA pol cannot transcribe at the same rate, which affects the number of transcripts that can be made from it
how can you tell the direction of transcription
NN pg. 2
find the consensus sequence - that is the coding strand which is transcribed from 5’ to 3’
answer practice questions on NN pg. 2
what is the primary energy source of E. coli
glucose
what happens when glucose is absent from the E. coli
E. coli expresses the genes of the Lac Operon to use lactose
B-galactosidase
an enzyme in E. coli that can break down lactose into monosacharides galactose and glucose
what gene expresses B-galactosidase
Lac-Z
describe Lac-Z
expresses B-galactosidase
- Lac-Z expression is inducible (lactose serves as the indcer for B-galactosidase expression)
draw the lac operon
NN pg. 3
operon
set of linked genes under the control of the same promoter
repressor protein
aka Lac I
binds to a segment of DNA called the operator
operator
a segment of DNA found between promoter and structural gene that acts as a control element
Lac Y
makes lactose permease –> allos the absorption of lactose
Lac A
encodes the enzyme galactosidase transacetylase –> breaks down a toxic compound that gets imported into the cell because of permease
linked genes
overall they are called structural genes
- Lac Z
- Lac Y
- Lac A
what happens when the repressor is bound to the operator and how does the repressor do this
OCCURS WHEN LACTOSE IS ABSENT
prevents transcription of structural genes
- Lac repressor works by looping DNA at the opperator (physically impedes RNA from progressing) –> prevents transcription
what happens when the repressor is not bound to the operator and how does the repressor do this
OCCURS WHEN LACTOSE IS PRESENT
lactose is converted to allolactose which binds to the repressor protein, which prevents the repressor from binding to the operator. This allows RNA pol to properly bind to its propmoter
negative regulation
when a repressor protein binds to the operator to prevent transcription
- repressed by the lac I repressor, which represses transcription when lactose is absent. this is regulated because it would be wasteful to transcribe when lactose is absent
polycistronic
lac operon mRNA is polycistronic, where the mRNA contains the coding sequence for the three different structural genes
what is the effect of a recessive-loss-of-function mutation on Lac I on the expression of the structural genes
NN pg. 4
they are constitutively expressed
what is the effect of a mutant whose operator is missing the key binding sites for the Lac I repressor protein
NN pg. 4
they are consitutively expressed
Lac I-
NN pg.5
Lac I never bound, repressor cannot bind to protein, Lac operon never expresed whether or not lactose is present
CONSTITUTIVE PHENOTYPE
Lac I+
NN pg.5
wild type
presence of lactose: lac operon expressed - inhibition relieved
absence of lactose: inhibits lac operon
can turn expression on / off, which means it has an INDUCIBLE PHENOTYPE
Lac I^s
NN pg.5
Lac I always bound whether or not lactose is present
UNINDUCIBLE PHENOTYPE
Lac O^c
NN pg.5
continual expression of the lac operon whether or not lactose is present
CONSTITUTIVE PHENOTYPE
Lac O+
NN pg.5
wild type
presence of lactose: lac operon expressed - inhibition relieved
absence of lactose: inhibits lac operon
can turn expression on / off, which means it has an INDUCIBLE PHENOTYPE
draw the answer: you found a mutant in E.coli that has a constitutive phenotype. how would you know whether it was Lac I- or Lac O^c mutation
NN pg.6
explain the answer: you found a mutant in E.coli that has a constitutive phenotype. how would you know whether it was Lac I- or Lac O^c mutation
NN pg.6
- each cell could be given a wild type copy of the Lac I+ gene and of the operator region (Lac O+) to make partial diploids (A partial diploid is a bacterial cell that contains two copies of some genes.)
IF…
Lac I- mutation, the wild type copy can make a repressor protein which can bind to the operator and work as normal
(INDUCIBLE PHENOTYPE)
IF…
Lac O^c mutation, the wild type copy can also make a repressor protein, but it doesn’t matter because the protein cannot bind to the O site, which causes it to continuously be made
(CONSTITUTIVE PHENOTYPE)
is a repressor cis or trans acting and explain what this means
NN pg. 6
trans
- wild type copy on the strand can recuse loss of function on other strand because its making a protein (proteins are soluble –> they can move around and repress their own operator or another operator)
is an operator cis or trans acting and explain what this means
NN pg. 6
cis
- wild type copy on the strand cannot recuse loss of function on other strand because they do not move - they control expression locally (same DNA)
haploid
Haploid refers to the presence of a single set of chromosomes in an organism’s cells.
diploid
(of a cell or nucleus) containing two complete sets of chromosomes, one from each parent.
consider a haploid E.coli LacI mutant with a dominant gain of function mutation in which it loses its affinity for lactose. no other portion of the protein has been effected. how does that affect expression of the structural genes
NN pg. 6
they become permanently inhibited (uninducible)
do practice problems on NN from pg 7 through pg. 9
positive regulation
NN pg. 10
- deals with activating transcription Lac operon activates transcription when glucose is absent. this occurs because its wasteful to transcribe the lac operon (lactose when glucose is present, because glucose is the preferred energy source for E. coli.
how does glucose regulate the lac operon
through cyclic AMP –> a cellular messenger that is a derivative of ATP
if you have high levels of glucose and low levels of lactose, how much cAMP do you have
low levels of cAMP (cAMP is inversely related to glucose)
if you have low levels of glucose and high levels of lactose, how much cAMP do you have
high levels of cAMP (cAMP is inversely related to glucose)
if you have low levels of glucose and low levels of lactose, how much cAMP do you have
low levels of cAMP (cAMP is inversely related to glucose)
if you have low cAMP how does that effect how much the lac operon is expressed
low levels of cAMP is correlated with low levels of lac operon expression
if you have high cAMP how does that effect how much the lac operon is expressed
high levels of cAMP is correlated with high levels of lac operon expression
adenylate cyclase
NN pg 11
resides near plasma membrane and functions to alert the rest of the cell when glucose is present / absent. it does this by converting ATP to cAMP. it only does this when glucose is absent
describe a working catabolite pathway when lactose is present and glucose is absent
NN pg 11
glucose absent –> ATP –> cAMP –> activate lac operon
how does cAMP / CAP complex activate the lac operon
NN pg. 11
cAMP creases, which allows for it to bind to CAP (catabolyte activation protein)
when cAMP is high, it can bind to CAP protein. when it binds, cAMP activates CAP and allows it to now bind to the region of the DNA called the CAP site.
the binding of the RNA pol at its promoter initiates transcription and activates the lac operon
answer practice problems on NN from pg. 12-13
