operons & restriction enzymes

how can enzyme activity be changed

• regulate activity of existing enzymes

  • positive/negative feedback regulation

  • inhibit/activate enzymes

• regulate the amount of enzyme

  • regulate “gene expression”

  • turn on/off genes that code for enzymes

trp in e coli

ecoli that lives in human colon needs amino acid tryptophan (trp) to survive

• get it by making it or from food a human eats

  • makes it through 5 step metabolic pathway

what happens if human host eats food with plenty of trp & effects

trp levels in colon rise — ecoli absorbs it & trp levels rise

1. trp inhibits 1st enzyme in methabolic pathway (negative feedback)

2. incresed trp levels causes genes that code for 5 enzymes in metabolic pathway to be shut off

operon

model for regulation of gene expression at level of transcription

• repressible: turn off

• inducible: turn on

model of operon

promoter then regulator gene then promoter region (where RNA polymerase binds & contains operator) then genes of operon

• regulator gene produces a repressor

trp operon

is normally on — RNA polymerase ecan bind to promoter & transcribe the 5 genes into mRNA

• will be switched off when trp repressor protein is activated & binds to the operator — needs a corepressor (tryptophan)

lac operon

is inducible — normally off (bc absence of lactose in environment, no need for the enzyme that breaks it down)

lac z gene

codes for beta galactosidase — breaks down lactose into usable sugars

how does lactose enter the ecoli cell

in the form of allolactose (isomer): less in milk so must be in significant amount to turn on gene

• allolactose binds to active repressor making it inactive — cannot block RNA polymerase

restriction enzymes

bacterial enzymes that recognize specific nucleotide sequences in dsdna & cleave the DNA at the site

• use as defenses against bacteriophages (virus)

cut piece of DNA at every recognition site — won’t cut its own DNA bc recognition sites will be modified

eco r1

recognition enzyme obtained from e coli

• cuts between GA indicated — must recognize the whole sequence first, don’t just cut at every GA

  • results in sticky ends

sticky ends

cut in staggered way — each side has short single stranded sequences on resulting restriction fragments

blunt ends

cut straight down middle — even length sequences on each side of restriction fragments

how can restriction enzymes be used to insert a gene into plasmid

1. obtain plasmid that has recognition site of specific re

2. cut out the gene from its genome using same re (need recognition sites for re either side of gene & gene needs same sticky ends as plasmid)

3. fuse gene fragment in (DNA ligase)

cutting a plasmid with re

• 1 restriction site = 1 linear piece of DNA

• 2 restriction sites = 2 DNA fragments

cutting linear DNA with re

• 1 recognition sites = 2 fragments

• 2 sites = 3 fragments

re digestion

dna is cut into different sized fragments using DNA

• can be separated & visualized using agarose gel electrophoresis (use stains for better visualization)

  • agarose acts as sieve (high [agarose] = smaller pores)

  • sorts the fragments by size