Bio Test 3b Flashcards
DNA ligase
joins DNA fragments in presence of ATP
What are the three vectors?
plasmid
viruses
aritificial chromosomes
plasmid
- extra chromosomal DNA molecule
- used for cloning small pieces of DNA
- has ori (replication orgin)
- selectable marker
- multiple replication sites usually with LacZ
ori
allows independent replication
selectable marker
allows presence of plasmid to be easily identified
sticky, overhang or preotruding ends
zig-zag cut by restriction enzymes where there is a protruding amnio acid on one side
palindromic sequence
sequence that is the same front and back
- ECOR1: GAATTC
- HindIII: AAGCTT
restriction enzyme or restriction endonuclease
- found in bacteria and archea providing a defense mechanism against invading viruses
- cut double-stranded DNA at specific spots
- 6 bp cutter (Sequence of 6 bp is isolated)
restriciton enzyme sites
site where restriction enzymes cut double-stranded DNA
artificial chromosomes
for larger insert and large-scale analysis of genomes
yeast and bacterial
when a gene is expressed what should it have?
ori
selection marker
multiples restriction sites
reporter gene
process of molecular cloning
- DNA cleavage
- ligation of gene into vector by DNA ligase in presence of ATP. recombinant plasmid made
- transformation
- screening
transformation
introducing recombinant DNA into bacteria
screening
uses selective medium containing antibiotics or X-Gal;
antibiotic
amplicillin; clones that contain the plasmid will grgow in medium with ampicillin
X-Gal
substrate for LacZ enzyme; clones that have the gene fragment inserted in LacZgene will turn white
transformation
DNA uptake by bacteria
transfection
DNA uptake by animal (eukaryotes) cell
transduction
injection of DNA into host cell by virus
conjugation
DNA transfer between two cells via direct contact
applications of molecular cloning
- expressing using proteins (insulin)
- expressing reporter protein (transgenic animals)
- pGLO plasmid
pGLO plasmid
contains GFP gene under the control of ara operator araC protein binds arabinose to form complex that turns on ara operon
process of DNA replication
- opening DNA double helix (helicase)
- building a primer (DNA primase)
- assembling complementary strans (DNA polymerase III)
- removing the primer (DNA polymerase I)
- joining okazaki fragments (DNA ligase)
characteristics of replication orgin
- spans 100-bp
- attracts intiator proteins
- has fewer hydrogen bonds and easy to open
- odten A-T rich stretches of DNA
- numerous in humans, one in bacteria
unique features of transcription in eukaryotes
- three RNA polymerase enzymes
- initiation complex forms at promoter with many transcription factors
- RNAs are modified after transcription
- One RNA makes one type of protein (monocistronic)
how do miRNAs regulate gene expression
- encoded by normal gene
- first cleaved into short dsRNA by DICER
- ss-miRNA forms complex with RISC
- siRNA pairs with target mRNAs
- causes mRNA degradation or blocks translation
what is the start codon?
AUG; Met
What is the only other codon that has only one representation?
Trp; UGG
What are the stop codons?
UAA
UAG
UGA
codon
every three nucleotides makes a code for amino acid
redundancy
some amino acids are encoded by multiple codons
ribosomes
- made of proteins and rRNAs
- consist of two subunits
- has 82 proteins and 4 rRNAs
tRNAs
- anticodon
- carries amino acid (attached to OH- group of three prime end of tRNAs)
- aminoacyl-tRNA synthetase
anticodon
3-nucleotide sequence on tRNA complementary to codon in mRNA
aminoacyl-tRNA synthetase
enzyme that attaches specific amino acid to corresponding tRNA
translation process
- initiation
- elongation
- termination
initiation
- small subunit of ribosome loaded with met-tRNA and initiation factors- complex
- initiation complex binds 5’ cap of mRNA
- initiation complex searching for first AUG codon
- large subunit binds
elongation
- new aminoacyl-tRNA binds to A site
- peptide bond forms by peptidyl transferase enzyme
- large subunit translocates
- small subunit translocates
- empty tRNA leaves ribosome
termination
- A site meet a stop codon
- release factor binds to A site
- dissociation of the translation machinery
antibiotics that kill bacteria by inhibiting protein synthesis
tetracycline
cycloheximide
tetracycline
blocks binding of aminoacyl-tRNA to A site
cycloheximide
blocks translocation of ribosome
translocation
location of ribosome
Where does protein synthesis begin?
on ribosomes in cytosol
signal sequences
- direct proteins to organelles
- 15-60 amino acids long
- usually removed after transport
- can be done through pore, across membrane and vesicles
transport by vesicles
from ER
golgi
endosomes or lysosomes
cell surface
import protein into ER the signal sequence will…..
have many hydrophobic amino acids
import proteins into the nucleus and signal sequence will….
have positively charge amino acids
