Biology 305 Exam 4

Translation

turning mrna script into protein

ribosome

assembly site for protein synthesis.

made up of large and small subunit.

rna+protein

function of small subunit

binding of mrna and initiation of translation

components of large subunit and function of each

a, p, and e site

a= recieves amino acid

p= growth of chain

e= exit

trna

short, folded rna molecule

has anti codon loop complementary to codon

creates mrna in 3’-5’ direction

functionn of aminoacyl trna synthetase

attach amino acid to the corresponding tRNA molecule

one per amino acid (20 total)

uses ATP as energy source

what starts initation in prokaryotes

Shine-dalgarno sequence

are prokarytes poolycisstronic or monocistronic

polycistronic (multiple transcriptions simueltanenously)

Transcription initation in eukaryotes

small subunit binds at 5’ cap and scans for first start codon (AUG)

are eukaryotes poly or monocistronic

monocistrnic (only one transccrption at a time via 5’ cap)

elongation process

scond trnna enters a site with helpp from elongation factor

chain transferred from fiirst to second trna, second trna moves into p site

first trna mves to e site and leaves

ribosome continues to move along sequence

what site does initaor trna enter at

p site

termination

stop codon does not have trna that is complimentary to it instead release factor binds which takes chain from trna and then releases chain

how many bases make a codon

3

can multiple different gene sequences code for same amino acid

yes

codon features

non overlapping

• single base mutation onnly affects 1 codon

triplet

• inserition and then deletion down sequence = normal reading past deletion point

non degenerate

• ore than 1 codon per amino acid

codon bias

different organism prefer differet codons for same amino acids

wobble rule

trna can recognize more than 1 gene sequence because 3rd base (5’ end) in trna can bind with more than 1 base, runs into issues when multiple different amino acid sequences bind to same codon

what proofreading goes on during dna replication

polymerase as 3’-5’ pproofreading abilities and exonuclease activity

every 10^5 there will be a base mutation

overall 1/(10^9) there will be an uncrrected base mutation during replication (very lo

post relplication repair in prokaryotes

methyl directed mismatch

old strand is methylated while new strand isn’t

loss of methylation at old strand were mutation is

creates mismatch error which is fixed by group of enzymes (muts-detector, mutl-liinker, muth-exonuclease)

once error cut out can be replaced by dna pol and ligase

new strand methylated

depurination

loss of nitrogenous base from nucleootide

deamination

amino group turs to carboxyl

oxiiddative damage

2 hydroxy groups become a carboxyl

clonal

mutation in all cancer genes

subclonal

mutation specific to a cancer

direct reversal

uv light damage causes photodimer (2 bases liinked together) which prevents basee pairing

cut out and reppaired

recoognized by base structure

base excision repair

recognizes and cuts wrng basse pair, Repairs damage to individual DNA bases. A specific DNA glycosylase recognizes and removes the damaged base, and the resulting gap is filled with correct nucleotides.

nucleotide excision

damage to double heix or multiple base pairs, cuts out nucleotides, creates new dna by bypass pol

translesion synthesis

dna pol stalls—> by pass pol replaced dna pol and inserts nonspecific dna sequences—>continuation of synthess—→ by pass pol falls off—> ppol 3 finishes synthesis

allows DNA replication to continue past sites of damage that would normally stall the replication fork

The bypassed lesion is often repaired by other DNA repair mechanisms after DNA replication is completed such as nucleotide or base excision repair

NHEJ

double strand break—> recognizes and repairs by just ligating two ends together

DNA by directly ligating the broken ends together without the need for a homologous template.

error free mutation repair methods

• mismatc

• direct reversal

• nucleotide and base excision

• homologous recombination

error prone repair methods

• translesion synthesis

• non homologus end joining chromosome

silent mutation

doesn’t change AA sequnce

missense

cahnges 1 AA

nonsense mutation

creates premature stop codon

inseriotn/deetion

inserting or removing 1 nucleootide causes frameshift

promoter mutation

• changes expression

• changes in transccription/translation

• unpredictable effects

northern blot

identify presence/absence of mrna sequence

western blot

identify presence/absence of protein

which ppolymerase is more prone to errors

by pass polymerase

Homologous recombination repair

highly accurate DNA repair mechanism that uses a homologous DNA template to repair damaged DNA, particularly double-strand breaks. precise repair of DNA breaks or gaps using an undamaged, homologous sequence (usually the sister chromatid

old school way of studying genes

generate random fragments using restriction enzymes—> clone by ligating into plasmids—> select for genes of intrest from the colonnies

modernn way

amplify genetic code via pcr→ replicated dna int plasmid

what does southern blot shhow

length of restriction fragments made by cutting up dna, presence/abseence of gene

what does northern blot show

intensity/revalence of gene detected

abundance and size of mrna (alernative splicing)

what does western blot show

identifies protein products

PCR left (forward) primer

5’-3’ drection same direction as leading strand

right (reverse) primer

3’-5’ reverse compliment of leading strand

PCR cycle steps

• denature DNA

• anneal pprimers

• extend from 5’-3’

• ampliifies dna between primers

reverse transcriptase pcr steps

• mrna reverse transcritio

• mrna+ cdna

• rnase degrades mrna leaving you with just cdna

• run cdna with normal pcr method

dideoxy sanger sequencing

• basic mechanism of dna relication is interrupted because the dideoxy nuceotides are missig two hydroxyl groups

• fragments of varying lengths

• run on gels

• read sequence from 5’-3’ top to bottom can then deduce 3’-5’ of coding strand

what is the preferred energy source for prokaryotes

glucose

lac Z function

produce b galactodose enzyme that breaks down lactose into glucose

lac y function

transort channel that allows influx of lactose into cell

lac A

functon more unknown thought to remove harmful byproducts of lactose metabolism

allosteric modificaion

lactose binding to repressor causes reversible conformational change that allows repressor to unbnd opperator region ad allows transcription of lac oeron to occur

wht is Iptg and what is it’s function

iptg is a non hydrolyzable lactose, can be bound by b gal but not broken down leads to constant high levels oof b gal activity

what is x gal

x gal is a lactose compound that pproduces blue byroducts when broken down. Measures activity of b gal

consttutive mutation

operon is always activated

uninducible mutation

operon is always repressed

cis mutation

ony affects gene expression on one dna sequence site, can’t be supplemented

mutation of dna binding sites

trans mutation

affects all gene expression sites in cell

suplementable

mutation of proteins

supper nducer (oc)

can’t bind repressor so operon is constantly activated

recessive

cis mutation

super repressor (Is)

can’t bind inducer so constant repression

dominant

trans mutation

catobolite repression

mediated by activator protein that binds to poymerase in absence of glucose that speeds up transcription or absence f lactose in the cell till glucose deleted

+glucose, - lactose

repressor binds ooperator

no induction

+glucose, +lactose

repressor not bound

activator not bound

low induction

-glucose, + lactose

repressor not bound

activator bound

high innduction

camp levels

low glucose= high camp

high glucose= low camp

reporter gene

protein encoding gene whose expression can be quantified/detected, couple it to gene of interest to drive/measure activity of gene of interest