Biochem exam 4 Flashcards
What are the functions of nucleic acids?
Storage and transmission of genetic information(DNA and RNA),processing of genetic information(ribosomes-catalytic mRNA),protein synthesis(tRNA, rRNA)
What are the functions of nucleotides?
Provide energy for metabolism(ATP), enzyme cofactors(NAD),signal transduction(cAMP)
What is the full name for DNA?
Deoxyribonucleic acid
What determines the amino acid sequence of proteins and nucleotide sequence of RNA molecules?
The nucleotide sequence in the cell’s DNA
What is the function of RNA
Carries genetic information from the site of DNA to the location of protein synthesis
Describe RNA
Components of ribosomes which is location of protein synthesis
What are the components of a nucleotide
a five carbon sugar,phosphate group,and nitrogenous base
What are the four nitrogenous bases present in deoxyribonucleic acids?
Adenine, guanine, cytosine, thymine
nucleotides are linked together by
dehydration synthesis or polymerization reactions between the sugar of one nucleotide and the phosphate group of another
Describe the sugar phosphate backbone
linkage of one nucleotide’s sugar and the phosphate group of another nucleotide
The strands of the DNA double helix are
antiparallel(opposite directions)
The two stands of DNA are held together by —–
hydrogen bonds between the nitrogenous bases of each strand
Cytosine bonds with
guanine
Thymine bonds with
adenine
What re the differences between DNA and RNA?
RNA contains the sugar ribose, the nitrogenous base uracil instead of thymine ,RNA is single stranded
Describe messenger RNA (mRNA)
Carry DNA primarily from nucleus to the ribosomes to serve as template for protein synthesis
Describe the function of transfer DNA (tRNA)
translates mRNA into a specific nucleotide sequence
What is the sugar found in DNA
beta-deoxy-D-ribosse
What is the sugar found in RNA?
Beta-d-ribose
What is a nucleoside?
A nucleotide without the phosphate group
What is a ribozyme
An RNA molecule capable of acting as a catalytic enzyme
What are the two types of nitrogenous bases present in nucleotides
pyrimidine and purine
What are the four nitrogenous bases in DNA
Adenine,Guanine, Thymine,Cytosine
What are the four nitrogenous bases in RNA?
Adenine,Guanine,Uracil,Cytosine
What makes the pyrimidine and purine bases?
The nitrogen contains lone pair of electrons which allows them to act as proton acceptors
Which are the pyrimidine bases?
Cytosine,Thymine, Uracil
Which are the purine bases?
Adenine,Guanine
Adenine pairs with?
Thymine in DNA and Uracil in RNA
Guanine pairs with?
cytosine
Thymine pairs with?
Adenine
Uracil in RNA pairs with?
Adenine
Cytosine pairs with?
Guanine
Cytosine pKa at N3 is
4.5
Thymine pKa at N3 is
9.5
Adenine pKa at N1
3.8
Guanine pKa at N7
2.4
Purines and pyrimidines are hydrophobic, and relatively insoluble in
Water at cellular pH
All nucleotide bases absorb UV light (strong absorption near —- resulting in electronic excitation
260nm
Do nucleic acids experience fluorescence?
no
The N-glycosidic bond is formed between the anomeric carbon of
the sugar in 𝛽 configuration and what position of the pyrimidine and purine?
N! of the pyrimidine, N9 of the purine
In nucleotides the pentose ring is attached to the base via a —- bond which is product of a — rxn
N-glycosidic bond, condensation
Can free rotation acoss the n-glycosidic bond occur in free nucleotides?
yess
The torsion angle about the N-glycosidic bond is defined by —- in purine
O4’-C1’-N9-C4
The torsion angle about the N-glycosidic bond is defined by — in pyrimidine
O4’-C1’-N1-C2
Angle near 0 degrees in the glycosidic corresponds to syn conformation
syn conformation
Angle near 180 degrees in the glycosidic bond corresponds to
anti conformation
the —- conformation of the glycosidic bond is most common in DNA
anti
Nitrogeneous base + Pentose + Phosphate and name ends in ylate
nucleotide
Nitrogeneous base + Pentose and name ends in osine
nucleoside
Epigenetic marker in prokaryotic cells
identifies own cell DNA to allow for destruction of foreign DNA
Epigenetic marker in eukaryotic cells
signals which genes should be active
Inosine sometimes found in the
“wobble position
” of the
anticodon in tRNA can be produced by —-
deaminating adenosine
Pseudouridine found widely in
tRNA and rRNA can be generated by
enzymatic isomerization of uridine after RNA synthesis
nucleotides are held together by
phosphodiester bonds
Why is RNA unstable?
single stranded, ribose makes it more susceptible to degradation
Hydrolysis of RNA is catalyzed by
RNases
How many H-bonds does a A-T form?
two
How many H-bonds does C-G form
three
Adenosine is part of many —— such as FAD and NAD
enzyme cofactors
What is the role of adenosine as a cofactore
provide binding energy and stabilize intermediates
What does FAD stand for?
Flavin adenine dinucleotide
What does NAD stand for?
Nicotinamide adenine dinucleotide
Some variations of nucleotide may serve as —- on the surface of cell prompting the production of a —-
second messenger
Some variations of nucleotide may serve as —-
second messenger
Inosine function
provides richer genetic code
Pseudouridine
stabilize tRNa and may help in folding of rRNA
primary structure of a nucleic acid
nucleotide sequence
secondary structure of nucleic acid
stable 3-D structure of a short polynucleotide segment(piece of DNA)
Tertiary structure of nucleic acid
folding of a nucleic acid into a chromosome or folding of large tRNA and rRNA molecules
A segment of 10 nucleotide pairs is approximately —- long
3.4 nm or 34 Angstrum
DNA is a —– —— helix
right handed double
the deoxyribose-phosphate back bone of DNA is
hydrophilic
nitrogenous base interactions are important for DNA stability and occur
perpendicular to the helical axis
The two strands of DNA run —- to each other
anti parallel
During DNA replication what occurs first?
Strand separation
Each separated DNA strand
serves as —- for synthesizing the new
strand based on ——-
a template, complementary pairing
Each new DNA Molecule consists of one — strand and one — strand
parent, daughter
Every 10 base pairs is a —- degree rotation in the DNA double helix
360
The form is the most stable
structure for a random sequence DNA
molecule under physiological conditions
B-DNA
Asymmetric attachment of the base pairs to the
sugar rings gives rise to
major and minor grooves
—– found in the major and minor grooves on the DNA helix can aid in determining sequence due to interactions with proteins
hydrogen bonding patterns
sequence specific interactions between DNA and proteins typically occur in the —
major grooves
sequence specific interactions between DNA and proteins typically occur in the —
major grooves
What are the three forms of DNA?
A,B.Z
Which type of DNA is left handed?
Z
how many base pairs per helical turn in the A form of DNA
11
how many base pairs per helical turn in the B form of DNA
10.5
how many base pairs per helical turn in the z form of DNA
12
What is the diameter of the B-DNA helix
20 angstrom
What is the helix rise per base pair in A-DNA
2.6 angstrom
What is the helix rise per base pair in B-DNA
3.4 angstrum
What is the helix rise per base pair in Z-DNA
3.7 angstrom
What is the glycosidic bond conformation for A-DNA?
anti (180 degree torsion angle)
What is the glycosidic bond conformation for B-DNA?
Anti(180 degree torsion angle)
What is the glycosidic bond conformation for Z-DNA?
anti for pyrimidines and syn for purines
A-DNA is favored in many solutions that lack
water
A DNA is a right-handed double helix, but
—— compared to B-DNA
wider
Is there evidence of A-DNA in cells?
no
Describe the shape of Z-DNA
left handed, thin, elongated
Z-DNA has a — shaped back bone
zig zag
Short stretches (tracts) of Z-DNA are found in
bacteria and eukaryotic cells
What are possible roles of Z-DNA
regulation of gene expression and gene recombination
Poly (A) tract
the appearance of four consecutive adenine in one strand may cause a slight bend in the DNA helix
Palindromic sequence
nucleic acid sequence in RNA or DNA that is identical to the complementary strand when they are both read in the same direction
What shapes may a palindromic sequence lead to
hair pin or cruciform shape
Mirror repeat
inverted repetition of a nucleotide sequence in a single strand that may lead to complex shapes
Hoogsteen DNA Structure
three strands . Two pyrimidine and one purine strand
G-quardruplex (G-tetraplex) DNA structure
four strands which can be parallel or two sets of anti parallel strands
What is the function of the G-quardruplex (G-tetraplex) DNA structure?
gene regulation, form a g rich seq
Why is single strand RNA right handed?
due to heavy preference of base stacking arrangement (typically purine to purine)
Self-complementary regions of ssRNA or dsRNA
tend to form
anti parallel A-form duplex
What is the wobble pair in RNA?
Uracil and guanine
How does a mismatched pair of nucleotides impact the secondary structure of a RNA/DNA
causes formation of bulges or internal loops
What is the tertiary structure of RNA
the formation of rRNA,tRNA, structure of ribozymes
H-bonds between —- contributes to the
folding of RNA into its
precise 3-D structure
non watson-crick base pairs
what are four examples of RNA tertiary
structure motifs
L-shape,A minor motif, ribose zipper, tetra loop
What is the difference between the base thymine and uracil?
Uracil is missing the methyl group at C5 of the pyrimidine structure
What allows RNA to experience some bonding interactions which are not possible for DNA
The presence of a hydroxyl group at C’2 of the ribose sugar allows for additional hydrogen bonds
Denaturation (melting) of double
helical DNA results in loss of?
loss of secondary structure in DNA and RNA
What are the most common causes of DNa/rna denaturization?
drastic changes in pH and extreme heat
How does denaturization affect the secondary structure of DNA/RNA
disrupts hydrogen bonding and base stacking between the base pairs
is denaturization reversible?
yes
what is renaturization?
return of pH and temp to ideal range to restore secondary structure
what does Hypochromism imply?
Absorption of light by nucleotides is reduced as it is renatured from single strand DNA to normal double strand helix
what is DNA melting temperature, Tm
temperature at which 50% of DNA is denatured and single stranded
Stability of DNA —— as the Tm increases
Tm
What factors impact Tm
DNA composition, DNA length, salt concentration
How does DNA composition impact DNA?
higher G-C content increases Tm due to more hydrogen bonds
How does DNA length influence Tm
Longer DNA has higher Tm
How does salt concentration influence tm
lower salt concentration means lower Tm
why does higher salt concentration increase TM
salt cations minimizes repulsion between the negatively charged phosphate groups
which methods can be utilized to detect a specific DNA molecule in a complex mixture?
radioactive detenction, fluorscent DNA chips
Which methods are useful in the amplification of a DNA sample
polymerase chain reaction, site directed mutagenisis
types of spontaneous mutagenesis
Deamination,hydrolysis of n-glycosidic bond, alkylation
what is deamination?
removal of NH2 group to C=O converting Ex: cytosine to uracil
What is hydrolysis of the glycosidic bond?
removal of nitrogenous base
What are some causes of DNA mutations
oxidation, UV light, ionizing radiation
What is included in all four reactions of the Sanger sequencing method
3’ to 5’ DNA template strand, DNA Primer, DNA polymerase, the four types of dNTP
What is different component in the four sanger reactions?
each reaction has a distinct ddNTP
What are the four steps in the synthesis of primer utilized in Sanger method of DNA Sequencing
Attaching the nucleoside to a silicia support, protectig group removed(DMT), new nucleotide added,oxidation to form triester, remove protecting groups from bases, removal of cyanoether groups from phosphategroups, cleavage of chain from silica support
How is single strand DNA obtained
Clone double strand DNA into plasmid configuration, inject plamsid into bacteria infected with phage, phage will close DNA in single strand
what is the purpose of PCR?
amplify target DNA by copying plasmids or areas of interest in linear DNA
what are the reaction components of PCR?
3’ to 5’ template of target DNA, primer, dNTPS, plymerase
What is the first step of PCR
denature template strand
What is the second step of PCR
annealing between the template and primer
Final step of PCR
Extension of primer via polymerase to produce complementary strand of template
What is recombinant DNA?
production of DNA sequence that does not occur naturally with the assistance of enzymes
Function of DNA endonucleases
cut a DNA sequence at a particular site
Function of DNA ligases?
paste
Function of polymerase
add or remove nucleotides from a DNA helix
How is DNA cloning technology utilized?
isolate and amplify the target gene from an organisms genetic material
General steps in DNA cloning
select vector to carry gene, PCR to amplify desired genetic material,introduce restriction enzymes to vector and PCR product to produce sticky ends, insert amplified gene into vector via DNA ligase, clone copies of host cells with added gene
Ori
origin point which directs replication in BAC
par gen in BAC
ensure even distribution of plasmid to daughter cells
CmR in BAC
gene expressing antibiotic resistance
lacZ gene in BAC
indicator of which colonies contained the inserted genetic material
In YAC Digestion with BamHI and EcoRI generates two separate
DNA arms, each with a
telomere end and selectable marker
Human DNA ligase utilizes
ATP
Bacterial DNA Ligase utilizes
NAD
what is the most common used DNA ligase
T4 DNA ligase
why is T4 ligase common in research?
Allows for the linkage of blunt and sticky ends in DNA
What is the required Co-factor for DNA Ligase?
ATP
Ligation is more effective with —— than —– ends
sticky ends, blunt ends
recombinant DNA is introduced into vector via — or —-
electroporation, or chemical transformation
Vector ligation in agar plate
growth of colonies but none contain inserted gene
vector + insert ligation in agar plate
growth of colonies with some containing inserted gene and other do not
insert self ligation in agar plate
no growth of colonies due to lack of antibiotic resistence
—– contain sequences that
allow inducible transcription of the inserted
gene
expression vectors
What is the purpose of expression vectors
to study the formation of protein from inserted gene
Definition of transcription
DNA is transcribed into RNA via RNA polymerase
Define translation which occurs in the ribosome
RNA sequence to protein
What are the parts of an expression vector?
promoter sequence, operator sequence, ribosome binding site, transcription termination sequence, multiple cloning sites,a selective marker
steps of protein purification
cell lysis to release protein,incubate w/ nicke-NTA beads,wash with salt solution,elute w/ imidizole competitor
during purification,Recombinant proteins with a tag bind
to
the affinity resin in purification column
How are tagged recombinant proteins released from the affinity resin in the purification column?
the addition of free glutahione solution
Which analytical techniques can be utilized to characterize a purified protein sample?
light scattering, crystallization , Raman spectroscopy , kinetics analysis
Eukaryotic genes contain — and —-
Exons and introns
Why can eukaryotic genes not be inserted into bacteria?
the bacteria does not have capacity to slice out introns
How can eukaryotic genes be inserted into bacteria?
transcription to mRNA which is intron free protein coding sequence
What feature distinguishes c-DNA from B-DNA?
cDNA does not contain introns but is still continuous and protein coding
What enzyme catalyzes the synthesis of mRNA into c-DNA?
reverse transcriptase
All mRNA molecules have a poly A tail what is its function
aids in extraction and purification of mRNA
-reverse transcription results in
a DNA/RNA hybrid
where the DNA strand is complementary to the
mRNA strand
cDNA is often used as template in to amplify genes
from eukaryotes
PCR
Membrane lipids have characteristic amphipathic structure
a hydrophobic tail and hydrophilic head
What molecules may be the back bone of a membrane lipid?
glycerol and sphingosine
What type of bond occurs between the back bone and head of a phospholipid?
phosphodieester
What type of bond occurs between the back bone and head of glycolipid?
glycosidic
What are the the three structures of membrane lipids in water?
micelles,bilayers,liposomes
Describe a micelle
Large round structure where the hydrophobic chains of fatty acids pointed towards middle with hydrophilic tails on the outer surface of sphere
what prompts the formation of lipids into micelles
concentration of lipid is above critical micelle concentration (cmc)
Describe the structure of a liposome
similar to lipid bilayer, but in a spherical shape
aqueous cavity in the interior
hydrophobic fatty acid chains of both layers point towards one another and away from the aqueous cavity
what is the purpose of a liposome
transport dissolved molecules in its aqueous cavity
different biological membranes have different —
composition of lipids and proteins
how thick is a typical the flexible biological membrane
50-80 angstrom
biological membranes for spontaneously in aqueous environments. What stabilizes these lipid structures
non-covalent structures such as hydrophobic interactions
biological membranes are permeable to — but impermeable to ——
small nonpolar/uncharged molecules, large polar molecules
Both proteins and lipids are free to ——– in the membrane
freely laterally
integral proteins are—– to the membrane
firmly attached
peripheral proteins are —- to the membrane
weakly bound to the and can easily be removed
the outer layer of the lipid bilayer is typically —-
more charged
are
usually located on the outer
membrane of the lipid membrane
glycolipids and glycoproteins
What are the types of membrane proteins
receptors, transportation,enzymes
purpose of of receptors on membranes
signal conversion from light, hormones, insulin receptors, neurotransmitters
function of enzymes on biological membranes
photosynthesis, atp synthesis
peripheral proteins are loosely associated to the polar head of outer layer of lipid bilayer via ionic interaction. What changes may disrupt these interactions to remove the protein?
change in pH or high salt concentration
what domains are common in integral proteins of membrane?
tm helixes and beta barrel motif
integral proteins are tightly bound to the biological membrane how can they be removed?
detergents which disrupt the membrane
Tyr and Trp are often found at the —– of the membrane
nonpolar/polar interface
the charged aa residues –,—,– are typically located in the —- layer of thhe membrane
lys,arg,his, inner
how many types of integral membrane proteins are there?
six
type II and I integral proteins have a single — and differ in the location of —–
transmembrane helix, the termini
type III integral proteins have ——- in a single polypeptide chain
transmembrane helixes
Type IV —– from
different polypeptides assemble
to form a –
tm domains, channel
Type V - proteins are held to the
bilayer primarily by
covalently bonded lipids
type VI has both —- and —
tm helices, lipid anchors
Amphitropic proteins describe attachment
covalently bonded to a lipid that is in the membrane
allow clustering of functionally related signaling proteins to produce a
highly localized and brief pulse of cellular signals
membrane rafts
what are the two phases of a membrane
gel like and fluid
describe the gel like phase of a membrane aka Lo (lipid ordered) state
individual lipids can not move
describe the Fluid phase aka lipid disordered state
(Ld phase)
the lipids can move
What causes the shift between the membrane phases
heat
Membrane fluidity is determined mainly by
fatty acid composition
what kind of fatty acid chains increase membrane fluidity?
short unsaturated fatty acids
Cell membranes of many
eukaryotes contain
sterols
Cell membranes of aerobic
prokaryotes contain
hopanols
lipids undergo —– within
the same layer via
Brownian motions
rapid lateral diffusion
Special enzymes such as —- catalyze transverse diffusion
flipasses
what is required for diffusion of lipids against membrane concentration gradient?
atp hydrolysis
Three types of phospholipid
translocators in the plasma
membrane
flippase, floppase ,scramblase
allows us to
study lateral diffusion by monitoring
the rate of fluorescence return
following photobleaching
fluorescence recovery after photobleaching (FRAP)
in single particle tracking,A single fluorescently labeled lipid molecule in a cell surface is
tracked on video by
fluoresecence microscopy
Lipids are organic molecules with —— soluability in water
low because they are hydrophobic
what are the four types of lipids?
signaling,storage, pigment,membrane
which lipids do not contain fatty acids?
hormones,cholesterol,pigments, vitamin a and d
which lipids do contain fatty acids?
membrane and storage
Standard nomenclature assigns C1 to the —–
and —– to the carbon next to it
carboxyl carbon , alpha
——- are essential nutrients
➢ Humans need them but can’t synthesize them
omega 3 fatty acids
The physical properties of fatty acids
are mainly determined by:
chain length and degree of saturation
Solubility of a —fatty acid as the chain length increases
decreases, increases
melting point of a fatty acid —- as
the chain length decreases
decreases and the chain length decreases
melting point of a fatty acid — as the number of double bonds increases
decreases
In vertebrates, free fatty acids
(unesterified fatty acids, with a
free carboxylate group) circulate
in the blood bound —–
to a protein carrier
covalently
However, fatty acids are present
in blood plasma mostly as
carboxylic acid derivatives such — and — which decreases their soluabilty
Biological waxes are esters of —-
long chain fatty acid with a long chain alcohol
purpose of waxes
protection,storage,waterproofing
Triacylglycerols are stored in —– as stored energy
and insulation.
fat cells
why are triglycerides better energy storage than carbohydrates
long term storage, more energy per carbon
what are the two types of phospholipids
glycerophospholipids
and spingolipids
types of Glycolipids
Galactolipids and spingolipids
structure of phospholipids
two fatty acid chains attached to glycerol. A polar group attached to hydrophilic head via phosphodiester bond
is the major component of most eukaryotic
cell membranes but not common in prokaryotes
phosphatidylcholine
role of phosphatidylcholine
blood platelet aggregation , plays role in inflammation
The backbone of sphingolipids is a long-chain
spingosine(amino alcohol)
in spingolipids the fatty acid is bound to spingosine backbone via —
amide linkage
Individuals with no active
glycosyltransferase will have the —- antigen
o
ppl w glycosyltransferase N-acetylgalactosamine group have the — antigen
A
Individuals with a
glycosyltransferase that transfers a
galactose group have the
B antigen
Sterol (structural lipids) have —- fused rings and a —– group in the A ring
4,hydroxyl
Steroids are oxidized derivatives of
sterols
Steroid hormones are
synthesized from —- in
— and –
cholesterol, adrenal glands and gonads
what is the function of steroids
signaling
Biologically active lipids are present in —- quantity than storage and structural lipids
lesser
What are the main biologically active lipids
signaling molecules, light absorbing pigments and lipid soluable vitamins (A,K,D,E)
