DNA footprinting/ operons/ trp operon 2-24/26 and 3/3

how does DNA footprinting work

radioactively label one end of DNA (usually 5’), put DNA in 3 tubes: one with sigma 38 BP, one with 678 and one with just DNA (control) add endonuclease with all and it will cut where there is no BP, then run a gel to see where protien is bound (footprint =missing/ faint region of DNA)

why are bacteria able to transcribe and translate a gene simultaneously

they don’t have a nucleus so the mRNA doesn’t have to be transported anywhere

what are the 3 necessary components of DNA foot printing

dsDNA, binding protein, endonuclease

what is polycistronic mRNA

single mRNA molecule that codes for more than one protein - in bacteria

what is an operon

in prokaryotes - group of genes transcribed together (promoter + genes)

how do bacterial ribosomes read proteins on polycistronic mRNA

jumps on the mRNA as it is being made and makes the proteins at the same time

waht does Lac stand for

lactose

what is the purpose of the Lac Operon

utilize lactose for energy, cuts lactose into allolactose or galactose and glucose (use when low levels of glucose)

why is the Lac operon important

it happens when low levels of glucose are present

what are the differnet parts of the Lac operon and thier function/ product

Lac O - operator message on/off of promoter, Lac z - makes β-galactosidase that cuts the lactose, Lac Y- makes gal. permease, channel for lactose to enter cell, Lac A - transfers acetyl group, stop the reaction, codes for protein galactoside transacylase

where does the repressor bind

lac o

whatdoes beta - galactosidiase do and why is it important to

it cutes the lactose into galactose and glucose or allolactose. needs to happen so glucose can be used for metabolism

where does the lac prespressor bind and how does this repress the gene

binds to the operator (lac O) and turns off

what encodes for the lac repressor

lac I

what does it mean when we say the operaon has leaky expression

you get some expression of products when repressor is on (represssor falls off occasionally, it also is moving from off/on)

What happens to repressors when not enough glucose and plenty of lactose is present

build up of allolactose, it binds to repressor, repressor falls of DNA and the RNA pol can bind and make mRNA

how does the level of glucose affect the lac repressor and expression of the lac operon

lots of glucose = lac pressor binds to operator and blocks RNA pol.

what does lac O do when repssor binds to it

it folds and makes a loop to close it off

what is CRP and what does it do after binding to cAMP

Camp receptor protein binds to the cyclic AMP which allows the CCRP to bind to the DNA at the lac promoter and the CRP helps the RNA pol. to bind better and start transcription

how does CRP binding to cAMP affect lac operon expression

expression = on and faster

what is the relationship between glucose and cAMP

inversly related, high glucose = low cAMP and low glucose = high cAMP

Describe the relationship between glucose and cAMP. Describe the different levels of Lac expression based on the different levels of Lactose, Glucose, and cAMP

high glucose, cAMP low and lactose present = low levels of gene expression , Glucose low cAMP high and lactose present = high levels of gene expression

what happens to expression if there is no lactose

cant pull repressor off so there is NO GENE EXPRESSION

what does the TRP operon do

syhtezies TRP

what controls TRP repressor binding to the operon promoter

high levels of TRP bind to repressor which bind to promoter which turns off operon

what kind of codon (what amino acid does it code for) is found on the regulatory region of the TRP operon?

.TRp A, TRP B, TRP C, TRP D, TRP E (→ EDCBA)

why would translation of regulatroy region 1 be slowed

because it needs TRP to run and so it has to fund the TRP that its making (region 1 codes for TRP so ribosome has to find TRP to make TRP)

when the ribosome is translating slowly on the TRP reglatory region what hairpin will form? what hair pin will form if the ribosome is traveling quickly?

slow = 2-3 hairpin (low TRP) fast = 3-4 hairpin (high TRP)

what happens when a 2-3 hairpin forms? what about a 3-4 hairpin?

2-3 nothing 3-4 = hairpin kicks pol and it falls off and excess trp binds to repressor and turns it off (rho independent termination)