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BIOL 107: EXAM 1
Watson and Crick |
- Reported DNA in 1953
- won nobel prize in 1962 and shared it with Wilkens
|
Nucleotide structure |
Nitrogenous base
5 C Sugar
Phosphate group |
Phosphate group |
One phosphorous atom bonded to 4 Hydrogen atoms
Negatively charged |
the connection between each nucleotide is a ... |
Phosphodiester bond
(phosphorous and 2 oxygens chemically bonded) |
3 Pyrimidines |
Cytosine
Thymine (DNA)- ethyl group
Uracil (RNA)- no ethyl group |
Characteristics of a pyrimidine |
6 membered ring that contains two nitrogen atoms |
Characteristic of a Purine |
hexagon+ pentagon combination |
2 Purines |
Adenine- N off the ring
Guanine- O off the ring |
Sugars |
DNA (deoxyribose) - H at 2' position
RNA (ribose) - OH at 2' position |
Chargaffs Rules |
%A = %T
%C = %G
%C+%T= 50% |
Watson and Crick model |
-Right handed double helix (from Rosaland Franklins X-ray)
-Sugar phosphate backbone outside
-Bases inside
-10 base pairs per turn
-strands are antiparallel
|
Base pairing |
A with T - forms 2 H bonds
G with C- forms 3 H bonds |
Macromolecules |
-Polymers formed from monomers-> great diversity
- fats and lipids are not polymers
- important for living organisms
- synthesis and breakdown, carried out with help of an enzyme |
Synthesis/Dehydration of macromolecules
|
Water is formed as a product for H and OH coming together
New bond created |
Breakdown/ Hydrolysis of a macromolecule |
Where H2O is added, breaking the bond into H and OH |
Enzymes |
accelerates chemical reactions but are not consumed by the reaction
- usually always a protein |
Proteins |
consist of C,H,O,N and S
- monomer repeat unit is an amino acid
- polymer state is a polypeptide
- can be found in folded structures |
Lipase |
enzyme that hydrolyzes ester bonds in phospholipids and triacylglycerides |
Amino Acids |
- 20 diff. AAs
- a carbon (central C connected to 4 diff. groups)
-4 groups (amino, carboxyl, hydrogen, R group)
- 19/20 naturally occurring
- Proline R group is attached to the N to create a 5 membered ring structure |
Amino Group |
N and 2-3 Hydrogens attached
- acts as a base (accepts a H and becomes + charged)
- at physiological pH (7) it is in a charged state with extra proton and +1 charge |
Carboxyl group |
-COOH
- OH group can act like a weak acid and give up a proton to solvent
- related to ketones and aldehydes |
Carbonyl group at physiological pH |
pH 7.2 in blood, carbonyl groups are deprotonated- they have a neg. charge associated with them |
Nonpolar amino acids |
Have R groups that are hydrophobic |
Polar amino acids |
Hydrophillic and have polar side chains
- second class
- interact with water through H bonding
- N and Q don't have side chains w/ acid/base properties b/c of carboxyl group |
Charged amino acids |
- electronically charged side chain, hydrophilic
- acidic AA have carboxylic side chains that deprotonate in aq. media near ph 2.7 (neg. charged)
- basis AA have side chains that are protonated at physiological pH and are + charged |
Polypeptide formation |
- occurs by dehydration reaction
- b/n carboxyl and amino
- loss of H20 forms C-N known as a peptide bond
-performed by ribosome
- analyze starting at N-terminus to C- terminus |
structure-function relationship of polypeptides |
not all polypeptides are proteins, must be folded to be a functional protein
structure dictates protein function |
Primary Structure |
determine shape and function, relies on covalent bonds
- list of AAs from N- to C- terminus
- single chain with peptide bonds |
Secondary structure |
- interactions b/n backbone groups
- H bonds
- a helix and b pleated sheets
|
Tertiary |
- interactions b/n R groups, not backbone of atoms
- 3D arrangement of secondary structures
- bonds include H, disulfide bridge, ionic bond, and Hydrophobic interactions
|
Quaternary structures |
- interactions b/n 2 or more polypeptides
- same interactions/bonds as tertiary |
AA polymerize to form protein.... |
a covalent bond forms b/n the amino group of one nucleotide and the carboxylic acid of the second |
Genes |
carry the information that encodes the traits that were observed in an organism |
chromosomes |
the chemical structures that contain genes, these are the molecules passed from parent to offspring during reproduction |
Griffiths experiment |
- early 20th century
- studied bacterial strains that cause pneumonia
- R strain- pathogenic (virulent), small and rough texture
- S strain- non-pathogenic (non-virulent), larger and smooth coat of polysaccharides
|
Griffiths 3 control reactions to experiment |
1. S cells into mice- died
2. R cells into mice- lived
3. heat treated S cells to make them non-living and observed the mice lived
* mixed heat killed S cells and living R cells and observed the mice died and pulled out living S cells |
Griffiths conclusion |
something in the heat killed S strain converted the live R strain to the pathogenic strain- a transforming substance |
Averys experiment- filtrate (1944) |
- new technology to hydrolyze biomolecules
- Started with S strain and generated filtrate of macronutrients
- complex sugars, fats, nucleic acids, and proteins |
Averys experiment- outcome |
treated 5 identical batches with one of 5 enzymes (RNase, DNase, Protease, Lipase, Carboase) and applied it to R strain bacteria
Outcome- DNase was the only one that transformed the nature of the S strain filtrate to |
Transformation (general facts) |
- uptake of DNA from environment
- single circular chromosome
- occurs when cell is stressed due to low nutrient density/ challenging agents
- 1% or less are competent (ability to take up exogenous DNA) |
gene expression |
creation of polypeptides from genes encoded in DNA- based chromosome
- DNA transcribed into mRNA
- mRNA translated into protein |
Location of transcription and translation in prokaryotes
|
both processes occur in the cytoplasm |
transcription and translation location in eukaryotes |
transcription occurs in the nucleus, translation occurs in the cytoplasm |
Central Dogma |
information flows from chromosome (DNA) -> RNA (mRNA) -> protein |
Triplet code |
4x4x4=64
only combination that codes for all 20 amino acids
singlet 4 =4
doublet 4x4=16 not enough |
codon |
3 nucleotides that specify one amino acid
ex. UGG= Trp
Exist in both DNA and RNA
|
Redundancy in genetic code |
- more than one code for an AA
- written as 5'-XYZ-3'
|
Start and stop codon |
start- start of the encoded protein sequence, usually AUG
stop- usually 3 at stopping point- UAA, UAG, UGA |
Template strand of transcription |
Hydrogen bond with incoming nucleotide pairs in RNA polymerase during synthesis
|
Coding strand of transcription |
- same sequence as mRNA product
- DNA sequence is ACGT instead of mRNA with ACDU |
3 stages of transcription |
initiation
elongation
termination |
Initiation |
- transcription begins at promoter
- promoter recognized by transcription factors and TATA box
- RNA polymerase begins to synthesize mRNA using the DNA template strand |
Elongation |
-RNA polymerase reads 3'-5' but writes 5'-3'
- doesn't require a primer
- uses nucleoside triphosphate as substrates
- writes AGCU but reads AGCT
|
Termination |
- RNA polymerase dissociates from the chromosome and releases the mRNA
- folds into a pair pin
- prok. involves termination sequences
- euk. are associated with RNA processing |
mRNA in prokaryotes |
- chromosomes and ribosomes in cytoplasm
- translation and transcription occur simultaneously in cytoplasm
- mRNA translated into protein as soon as it is made |
mRNA in eukaryotes |
-mRNA made in nucleus, ribosomes in cytoplasm
- transcription and translation can't occur at the same time
- mRNA transported from the nucleus to the cytoplasm |
mRNA processing in eukaryotes |
- pre-mRNA (in humans, plants, and yeast) is made during transcription
- pre-mRNA modified into mRNA in nucleus |
RNA processing in eukaryotes |
5' end capping- add. of a methylated guanine (protects the end)
3' end capping- add. of poly A tail
UTR- untranslated regions, one next to 5' cap, the other next to 3' tail |
Function of cap and tail |
1. mRNA translation
2. mRNA export from nucleus, no export of cap or tail are missing- Quality control
3. mRNA stability in cytoplasm- when poly A tail degrades, the mRNA is removed from the cell through hydrolysis |
RNA splicing in eukaryotes |
- removes "intervening" sequences (Introns), not protein coding
- unites "exported" seq. (Exons) b/c protein coding
- this produces mature protein coding seq. in translation |
RNA splicing |
- branch site in an intron is a specific RNA seq. 5'-UGA or UAA
- branch site attacks the donor site at the 5' end of the intron, creates lariat structure
- 3' end of upstream exon attacks the acceptor site in the nearest intron
- connects 2 eons and leads to release of intron which is hydrolyzed |
Gel electrophoresis of RNA |
- focus on size and length
- neg. charge at wells, pos. at far end due to neg. charge of phosphate group in RNA |
importance of gene regulation in eukaryotes |
-contain control elements
- seq. that regulate transcription, upstream of promoter
- enhancer region, independent transcription binding sites, upstream of the gene
- proximal control elements close to 5' end to contain multiple transcription factor binding sites |
General Transcription factors |
used for all protein-coding genes, recruits RNA polymerase
- bind to proximal control elements of almost all genes
|
specific |
used for a set of protein-coding genes
- req. for high levels of transcription
- both DNA and other regions that bind to proteins |
Transcription activation facts |
-occur over long distances
-DNA bending proteins assist- enhancer and proximal control regions neighbor |
order of transcription activation |
1. activator (tr. factors that increase gene expression) bind to the enhancer region of the gene
2. general tr. factors bind to proximal control region
3. mediator proteins- bind to both the activators at the enhancers and the general transcription factor at the proximal control region |
transcription repressors |
-repressors bind to sequences in the enhancer region called silencers to turn off gene transcription
- DNA bending proteins & mediator proteins are needed |
Cell differentiation |
activators and repressors differentiate cells with the same genomic DNA |
RNA interference (RNAi) (discovery) |
Fire & Mello- RNA silence-gene expression in eukaryotes, won the nobel prize |
Translation is RNA mediated |
mRNA
rRNA
tRNA |
What does tRNA do in transcription |
brings specific AA to ribosome and inserts the AA into the growing polypeptide chain if the correct codon is found in mRNA |
tRNA features |
- clover leaf structure- base pairs stabilize structure
- folds further into L shape
- anticodon: base pairs with mRNA codon |
tRNA charging |
-AA attached to the opp. end of the tRNA from the anticodon (3' end)
- only one type of AA is attached to each tRNA= makes tRNA charged when bound |
function of ribosome (general) |
-rRNA plays a catalytic role = protein synthesis
- ribosomal proteins hold rRNA, mRNA, and tRNA in place to carry out protein synthesis |
specific functions of ribosome |
1. bind to mRNA to read codon w/ high specificity
2. bind ot tRNA so mRNA is read w/o errors
3. catalyze peptide bond formation b/n incoming AAs and the growing polypeptide chain
4. ribosome must move along the mRNA so it can read each codon in order |
subunits of ribosome |
large- has all tRNA binding sites
small- contains mRNA binding site |
Binding site of ribosome (large subunit) |
A site
P site- peptidyl
E site |
general steps of translation |
initiation: protein synthesis
elongation: adds AA to generate a longer polypeptide chain
termination: ends protein synthesis when all of the coding region is read |
Initiation of translation in prokaryotes |
-small subunit binds mRNA
- use ribosome binding sequence so the start codon is lined up with the start site of translation
- initiator tRNA binds start codon (AUG), charged with AA Met
- large subunit joins small subunit, initiator tRNA is at P site
- energy from conversion of GTP to GDP |
Initiation of translation in eukaryotes |
-only a single protein coding region
- loading or ribosome occurs at the 5' cap of mRNA
- scans mRNA toward the 3' end till it reached the 1st 5' AUG seq.
|
Advantages of Prokaryote translation |
anywhere there is a ribosome binding sequence, the following protein can be synthesized, bacteria can increase expression of related genes |
Elongation of translation |
1. recognition of mRNA codon in the A site by nuclease pairing to the correct charged tRNA
2. form. of peptide bond b/n the amino group of the new AA and the c-terminus of the growing peptide chain
3. translocation of the newest tRNA from the A site to the P site (5'-3') |
Codon recognition of elongation in translation |
aa-tRNA binds A site
tRNA anticodon base paires to the mRNA codon present in the A site |
Translocation in elongation of translation |
-peptidyl- tRNA moves from A to P site
- empty tRNA moves from E site- gets released
- new mRNA codon is exposed at A site, ready for new aa-tRNA
- cycle is repeated until a stop codon enters the A site |
Peptide bond formation in elongation of translation |
- bond b/n new AA at A site and polypeptide at P site
- transfers polypeptides to A site
- catalyzed by RNA |
Termination |
-release factors bind stop codon in A site- cause hydrolysis that releases polypeptide
- subunits can dissociate |
Protein misfolding diseases |
-Scrapie
- Parkinsons Disease
- Alzheimers
- Transthyretein |
Mad cow disease |
introduction of one misfolded protein can stimulate the production of misfiled copies
|
Chaperonin proteins in the cell |
1. unfolded polypeptide enter the cylinder from one end
2. cap attaches- cylinder changes shape, creating hydrophillic enviro. for folding
3. cap comes off and folded protein released |
Gel electropheresis, studying proteins in primary structure |
- GE separate proteins by size instead of nucleic acids
- SDS-PAGE gel used
- protein size measured by molecular weight in kiloDaltons
- proteins are unfolded by "SDS" before ran through the gel- must be primary structure |
mutations are... |
changes in the DNA sequence that give rise to new alleles and can be passed down |
Point mutation |
a change in one base pair
ex. THE CAT
SHE CAT |
sickle cell anemia |
a point mutation, red blood cells clump and clog the vessels
Causes changes in the 6th AA of the primary structure = changes in quaternary structure
|
3 types of point mutations |
- substitution
- insertion
- deletion |
substitution mutation |
genes found in 2 individuals differ at one location
ex. GGAG
GGGG |
Insertion mutation |
one copy of a gene will have more nucleobases as compared to the wild type sequence
ex. CTGGA
CTGTGAGA |
Deletion mutation |
opposite of insertion, a gene is missing one or more nucleobases
ex. CTGGAG
CTAG, GG missing |
Silent |
a type of substitution
- no change in the amino acid
- changes happen at the 3rd base of a codon |
Missense mutations |
change in the AA
- changes at 1st/2nd codon
- sickle cell anemia |
nonsense mutations |
-changes to a stop codon
- neg. mutation because a truncated protein will be produced
- shortened polypeptide is not functional |
How do mutations arise |
- spont. changes in DNA- chromosome
- induced changes due to mutagens (UV light or ionizing radiation)
- polyaromatic hydrocarbons cause mutation (in cigarette smoke) |
Frameshift mutation |
change in nucleotide # not in a multiple of 3
- mRNA changes which creates a new protein sequence
|
Non-frameshift mutations |
change in nucleotide # in multiple of 3
- entire codons lost/gained
- resulting protein will be shorter/longer by a total of N/3
- N = # nucleobases added/lost |