biochem exam 1

definition of Tm

melting temp of DNA

• 50% of dsDNA and 50% of ssDNA

A260

measures absorption

• based on pi electrons and aromatic

temperature and absorption

temperature increases —> bonds break and base stacking weakens —> pi electrons loosen —> able to absorb UV

• HIGHER TEMP = MORE ABSORPTION

factors that affect Tm

1. GC Content: higher GC = higher Tm

2. Salt/Ionic Strength: more cations = higher Tm

• neutralize the negatively charged phosphate backbone

1. DNA Length: longer DNA = higher Tm

holiday junction

4 dna strands intertwined

benefits of holiday junction

genetic diversity

antisenseoligonucleotide

• single stranded DNA or RNA

• MOA: binds to target mRNA —> activate RNases, steric hindrance = trnaslation inhibition, exon skipping or inclusion

  • bind to miRNA —> inhibit miRNA

siRNA

double stranded RNA

MOA:

1. into endosome

2. long strand cleaved into siRNA by dicer

3. siRNA enters RISC —> one strand is discarded and other is kept —> guide strand binds to complementary —> mRNA degradation

• degraded by ribonucleases

miRNA

double stranded RNA

1. cell's DNA is transcribed into a RNA molecule with a hairpin loop shape, called pri-miRNA.

2. Drosha cuts RNA into a smaller hairpin loop called pre-miRNA.

3. to cytosol

4. Dicer chops off the hairpin loop, leaving a short, double-stranded miRNA

5. enters RISC, One strand is discarded and degraded and other strand is kept.

6. guide strand binds to a target mRNA —> suppression of transciption or mRNA degradation

importance of psuedouridine in vaccines

1. decrease immune response to vaccines

2. nancanoical = more stability

CRISPR cas 9 works on what level

DNA

CRISPR CAS 9 effect

permanenent bc affecting genetic material

ways to do DNA footprinting

1. hydroxyl radical

2. protein/ligand bound

protein/ligand bound

1. fluorance label one end of the DNA

2. add DNA with the protein of interest

3. add cleavage agent at low concentrations so each DNA molecule is cut average once.

4. Where protein is bound, it protects the DNA from cleavage.

5. Run gel —> the missing bands indicate the precise sequence where protein was bound

hydroxyl radical

1. fluorescent label one end of DNA

2. Mix your labeled DNA with your protein of interest

3. radicals attack the accessible DNA backbone

• hydroxyl group binds to hydrogen —> free radical

1. Missing lane reveal the exact nucleotides where the protein shielded the DNA from the radicals.

transcription steps

1. intiation

2. elongation

3. termination

transcription direction

template strand: 3 —>5

coding strand (what you copy)/synthesis : 5 —> 3

transcription uses…

rNTP

RNA vs DNA polymerase

RNA cannot pop on/off

• remain attach to template until termination

RNA structure levels

1. primary

2. secondary

3. tertiary

** secondary faster than tertiary

bacteria and eukaryotes transcription

bacteria: transcription + translation can happen simulatenously in cytoplasm

eukaryotes: transcription in nucleus —> translates in cytosol

A minor

stabilize tertiary structure

• on minor groove

• hydrogen bonding and van der Waals with unpaired adenine bases

eukaryote vs prokaryote

euk: splicing out introns/exons; slower

• use splicesome

code degeneracy

muliple codons code for one amino acid

• usually at 3rd nucleotide (wobble)

A, P, E sites

1. A = aminoacyl site; tRNA enters carrying amino acid

2. P = peptidyl site; peptide bond form here

3. E = exit site; release polypeptide

water exclusion

nonpolar/hydrophobic aggregate

structure of amino acid

1. Amino group

2. Carboxyl group

3. Hydrogen atom

4. Distinctive Side Chain

how peptide bond formed

leaving group = water (condensation)

add water = hydrolysis

frameshift

skipping one nucleotide

energy during translation (euk vs pro)

1. EFTU = bacteria; interacts with GTP

2. ATP

3. GTP hydrolysis = both euk and pro

tRNA shape

2D = cloverleaf

3D = L shaped

why tRNA shape important

• One end contains the anticodon

• other end contains the 3′ CCA acceptor stem, where the amino acid attaches

how do tRNA work

1. An aminoacyl-tRNA synthetase attaches the correct amino acid to tRNA.

2. The charged tRNA enters the ribosome's A site.

3. Its anticodon pairs with the mRNA codon

4. The growing peptide is transferred to the amino acid on the A-site tRNA.

5. The ribosome moves forward, shifting the tRNAs through its binding sites.

What happens to A1492 and A1493 during translation decoding?

• A1492 and A1493 are adenine residues in bacterial 16S ribosomal RNA.

• they flip outward from their normal positions

• They interact with the codon–anticodon minihelix to help verify correct base pairing.

decoding

ribosome checks whether the tRNA anticodon correctly matches the mRNA codon.

How does paromomycin affect protein synthesis?

def = promote miscoding by stabilizing codon-anticodon pair even if not correct

• stable A minor interaction bw ribosomal RNA and tRNA-mRNA

TRANSLATION ERRORS

How does puromycin affect protein synthesis?

DEF: Resmbles 3’ aminoacyl end of a charged tRNA

• lack structure to do elongation —> released

STOPS PROTEIN SYNTHESIS EARLY

L vs D

COO ON TOP AND R GROUP ON BOTTOM

NH2 on left = L

on right = D

most amino acids are..

L forms except glycine

aliphatic

carbon and hydrogen

peptide orientation

always N —> C

N terminus = with free amino group (NH3) - positive

C terminus = free carboxyl group, COO - negative

peptide bond rotation

• phi = N — C alpha

• psi = C alpha — C

CANNOT

C — N

alpha relix calculation

1 turn = 3.6 amino acid = 5.4 A length

beta sheet types

1. Parallel = Adjacent strands run in the same N-to-C direction

• less stable H bond (HHHH)

• favors inside

1. Antiparallel = Adjacent strands run in opposite N-to-C directions

• stable H bond (HPHPHPHP)

• one side inside and other side faces solvent

beta turns and amino acids

when the protein chain needs to change direction

glycine and proline

alpha helix prefer

small or charged

beta prefer

large nonpolar or aromatic aa

alpha vs beta

alpha

1. spiral

2. h bond within helix

3. side chain point outward

beta

1. pleated sheet

2. h bond between strands

3. side chain alterate above/below

tertiary stabilized by

1. hydrogen interactions and hydrogen bond

2. ionic interaction (salt bridges)

3. van der waals

4. disulfide bonds

post translation mod

modifications to a protein after translation

types of post translational modification

1. phosphorlyation = add phosphate

2. glycosylation = add sugar

3. acetylation = add acetyl

4. methylation = add methyl

5. ubiquitination = ubiquintin protein

6. disulfide formation = covalent S — S bond betwen cysteines

structure of ribiosome and fxn

one small subunit and one large

small = decode mRNA

large = catalyze peptide bond

disulfide bond formation

Two cysteine thiol groups (–SH) undergo oxidation to form a covalent S–S bond

• common in ER

translation steps

1. Charged tRNA enters A site

2. Correct codon–anticodon pairing is checked

3. Peptide bond forms, transferring the growing chain to A-site tRNA

4. Ribosome translocates one codon along mRNA

5. A-site tRNA moves to P; P-site tRNA moves to E.

6. Empty tRNA exits; cycle repeats

direction of ribosome

moves 5′ → 3′ direction

RNase A

enzyme that breaks down RNA by cleaving phosphodiester bond

protein dialysis

removing small molecules from protein solution via semipermeable membrane

What are common protein-denaturing conditions?

● High temperature

● Extreme pH

● Urea

● Guanidinium chloride

● Certain detergents

denaturing agents affect which structure

seconday, tertiary, quatenary

reducing vs denaturing

denature = disrupt folding interactions

reducing agent = break disulfide bonds

BOTH RETAIN PRIMARY

LOCK AND KEY

enzyme and substate fits perfectly

induced fit

subtrate bidning —> promote conformational change —> improve enzyme fit

when can recover structure

1. denaturing alone

2. denature + reduce —> removed

is protein folding random

NO

• guided by energy

What is Levinthal's paradox?

proteins fold too fast for folding to be random

Explain the free-energy funnel model

unfolded ensemble = many confromations possible

native state = fully folded, lowest energy conformation

model = free energy decreases as the protein approaches its native state

types of enzyme inhibitors

1. competitive = compete with substrate for active site

• icnrease KM same Vmax

1. pure noncompetetive = binds E and ES equally at another site

• same KM lower Vmax

1. uncompetitive = binds on ES complex

• lower Km and Vmax

1. mixed = bind E and ES with different afffinities

• increase/decrease Km and lower Vmax

Vmax

max reaction velocity when enzyme active sites are saturated with substrate

Km

substrate concentration where velocity is half of Vmax

competitive vs pure noncompetitive

curve change

1. comp = shift right

2. noncomp = lower plateu

more substrate helps

1. comp = yes

2. non = no

Metal concentrations and their safety in the human body depend heavily on whether they are in a ___ or ___ state 

OXIDIZED / REDUCED or free/bound

• free = more available to react

dominant cation outside cell

Na

dominant inside cell

K

major noncovalent bond

1. hydrogen bond - specificity

2. van der waals - stability of protein/nucleic acids/membrane

3. electrostatic interactions

4. hydrophobic interactions

what influences solubility

polarity

peak in energy diagram

transition state - activation energy

• reactants - peak

enzyme catalyze rxn how

lower activation energy

thermodynamics vs kinetics

kinetics =how quickly rxn is

thermodynamics = determine whether rxn is favorable and where rxn ends uo

• direction, spontaneity

enzymes affect what

kinetics

binding affinity and gibbs free energy

higher binding = more negative gibbs

life characteristics

1. self maintentance

2. reproduction

3. evolution

DNA —> DNA

DNA —> RNA

RNA —> Protein

RNA —> DNA

1. replication

2. transcription

3. translation

4. reverse transcription

nucleotide components

1. nitrogenous base

2. 5 carbon sugar

3. phosphate

pyrimidines and purines

pyrimidine = 2 rings

• UTC

pyrine = six membered ring

• AG

separate DNA method

gel electrophoresis = separate DNA via size and charge

• bigger size = slow

• strength of E field determines how fast

• positive side at end - DNA is negative

common DNA

B form

force keep DNA stable

base stacking

Major and minor groove

minor groove = replication fidelity and non specific binding

• shape recognition

major groove = sequence recognition, sequence specific binding proteins

• look at hydrogen bond donors/acceptors and methyl groups

nonconventional base pairings

1. wobble

2. hoosten

pros of synthetic polymers

1. different chemical properties

2. greater stability

3. increased resistance to degradation

4. novel structures

solubility of phosphate, pyrophosphate, cyclic-trimetaphosphate

phosphate > pyrophosphate > cyclic trimetaphosphate

Unhappy form

DNA polymerization rxn

reactants

- DNA strand/primer 

-dNTP 

-Mg and polymerase

Product: 

• DNA strand one nucleotide longer 

• pyrophosphate (PPi) released

DNA polymerase shape

hand

• fingers = binds incoming dNTPs and template

• palm - contain catalytic site with divalent metal ion

• thumb = hold DNA

metal ion for polymerase catalysis

Mg2+

Enzymatic DNA synthesis (or EDS) uses a specialized protein called?

terminal deoxynucleotidyl transferase (TdT)

• template indepdent

• add nucleotides to strand without template

first generation

sanger sequencing

second gen

sequencing by synthesis

third gen

single molecule sequencing

when do disulfide break and form

form: oxidative

break: reduced