Molecular Bio (Lectures 1-7) Exam 1 Prep

what is a gene

stretch of DNA that codes characteristics

what is an allele

variants of gene (changes in code)

what is an ORF

stretch of DNA (start to stop codons)

what is a mutation

process of change

what is variation

result of mutation

what is the defining chemical characteristic of RNA

2’ hydroxyl in addition to 3’ hydroxyl

what is the structural difference between purines and pyrimidines

purines have 2 rings while pyrimidines have only 1 ring

what is this base

adenine

what is this base

guanine

what is this base

cytosine

what is this base

uracil

what is this base

thymine

what is the key difference between nucleotides and nucleosides

nucleotides are nucleosides with a phosphate group

what is the bond that attatches phosphate groups to nucleosides

phosphodiester

what is a nucleoside

pentose with base (can be ribose or deoxyribose)

what is a nucleoside + 1 phosphate called

nucleotide monophosphate

what is a nucleoside + 2 phosphates called

nucleotide diphosphate

what is a nucleoside + 3 phosphates called

nucleotide triphosphate

what are the 5 nucleobases

guan, thym, aden, cyt, and ur (5 base pairs)

what is the nomenclature of a nucleoside

base + sugar (-osine for purines, -idine for pyrimidines)

what is the nomenclature of a nucleotide

base + sugar + #phosphates

what end are polynucleotides built upon

3’ free OH

how many base pairs/turn are in DNA

10 base pairs per turn

what groove of DNA has the most contact with other molecules

major

what is the total percentage rang of G + C

22 to 73%

what does a high content of GC pairs indicate

higher heat tolerance

what does a low content of GC pairs indicate

lower heat tolerance

why does absorption increase as temperature increases

as temp increases, h bonds break which allows for more surface area to interact with UV rays

what is melting temperature

the temperature at which DNA strands are ½ denatured

why does human DNA have a more complex heating curve

repetitive DNA allows remaining DNA to find each other faster

besides DNA, what can also denature DNA

organic solvents, high pH, and low salt concentration

why does GC content directly (and linearly) affect DNA density

GC has a larger molar volume than AC base pairs

what is annealing/renaturation

after DNA separation, 2 strands come back together (under proper conditions)

what is the best temperature for annealing

25 degrees Celsius below Tm

should there be higher or lower DNA concentration to best suit annealing

higher concentration (within limits)

is renaturation time important?

yes! as time increases, annealing increases

what is hybridization

process of putting together a combination of two different nucleic acids (1 DNA strand + 1 RNA stand or 2 DNA strands or 2 RNA strands with complementary/nearly complementary sequences)

what is the mw of 1 base pair (average of 2 nucleotides)

660 Da

what is the mw of 1 base of RNA

abt 350 Da

what can electron microscopy estimate about DNA

its size and shape (circular vs linear)

what is the mw of the average protein

40,000 Da

what is the average mass of an amino acid

110 Da

how many amino acids does a protein contain on average

364

how large is an e.coli chromosome

abt 4200 proteins

how large is Phage I

abt 44 proteins

how large is Phage II

5 proteins

what is c-value

DNA content per hapoild cell

what is the c-value paradox

caveat based on repetitive DNA and non-coding regions that states more complex organisms will not always contain proportionate increase in genes

What will differ about multicellular organisms with similar amounts of genes

differing amounts of base pairs

in what is it possible for RNA to exist as complete double strands

viral genomes

what does a bulge in RNA indicate

addition of 1 nucleotide

what does an internal loop in RNA indicate

addition of 2 nucleotides

What does a hairpin in RNA indicate

looped RNA

why do aromatic amino acids absorb UV wavelengths

conjugated pi systems give electron transitions of the right energies

what constitutes a eukaryote

presence of nucleous

how does one isolate biomolecules

1. 1. cell LYSIS

2. 2. protein denaturation/digestion to PURIFY

3. 3. alcohol PRECIPITATION (makes DNA into pellets)

how does one stimulate DNA/RNA binding in silica isolation

make bridge using intermediary Na+ ion and wash (takes place of H2O)

what would be a good wash solution to get rid of salt in silica isolation

solution with high concentration of salt and little organic material

what is the elution going to require in silica isolation

water to take off Na+ and carry DNA with it

what happens to RNA at lower pH

purines get cleaved off

what happens at higher pH

RNA cleaves itself off through oxidation

what are the most common methods of protein purification

precipitation, column chromatography, and gel electrophoresis (for analysis, not purification)

what defines an analytical technique

the use a small amount of protein that gets modified or destroyed in analysis

what defines a preparative technique

a large amount of protein that maintains native protein activity

what are examples of protein precipitation

ammonium sulfate and EtOH

what percentage of protein solution becomes soluble during protein precipitation

40%

at what percentage does a supernatant become insoluble

50%

by what is solubility affected

pH and ionic strength (salts)

what occurs during size exclusion chromatography

small beads stay inside pores to filter protein by size

what elutes first in size exclusion chromatography

larger proteins

in anion exchange, is the pka higher or lower

higher (abt 11 pka)

in cation exchange, is the pka higher or lower

lower

what is inside ion exchange chromatography

polymer beads with negatively charged functional groups

in cation exchange, what elutes faster

proteins with a more negative charge

in anion exchange, what elutes faster

proteins with a more positive charge

what is inside affinity chromatography

polymer-bound ligand specific for POI

in affinity chromatography, of what does the elution consist

imidazole ions from histadine

what method allows for direct visualization of DNA and RNPs

electron microscopy (TEM and cryo-EM)

what methods allows for indirect visualization of nucleic acids

gel matrices, fluorescence

what does the presence of fractions on a band indicate

they have the desired material and appear due to the radioactive label

what procedure detects DNA

Southern blot

what procedure detects RNA

northern blot

what procedure detects proteins

western blot

how do DNA/RNA blot techniques work

radioactive complementary strands

how do protein blot techniques work

naturally occurring protein-protein interactions - antibodies generated by injecting a small animal with the protein you wish to follow - after a few weeks to months, the animal produces antibodies against your protein

what does the radioactively labeled secondary antibody attach to in western blotting

a primary antibody

what shape are all eukaryotic chromosomes

linear - capped by telomeres

how many base pairs of DNA exist in each of our cells

6 billion - abt 6ft long

how small much the DNA compact into to fit inside the nucleus

10 microliters

what charge does a histone core carry

positive

what is a nucleosome

histone core protein + dna

what is the minor “linker” histone called

H1

what are the 4 major histone proteins inside the core

H3, H2B, H2A, H4

how many variants of H4 have ever been reported

2

how many bp of DNA are in a single nucleosome

147bp

how many times does DNA wrap around histone

1.67times

what is a prokaryotic chromosomal DNA replication called

catenane

what is the function of topoisomerase 1

relax positively supercoiled DNA

on what cofactor is top1 dependent

ATP

what is it called when one phosphodiester bonds breaks and one phosphodiester bond is made

isoenergetic