Wave 12—CH14 END OF EXAM 2 CONTENT

Lac operon historically

First cluster of genes that taught us how genes are regulators

Operon

Cluster of genes under the control of a single promoter. This is a prokaryotic thing.

polycistronic mRNA

MRNA that can produce more than 1 protein

Monocistronic mRNA

One mRNA —> one protein

Do eukaryotes have polycistronic mRNA?

No, they want finer control over gene expression.

Inducer

Small organic molecules that function nas signals. When it binds it causes transcription by causing the repressor to lose affinity to DNA and stop repressing transcription.

Co-repressor

Small organic molecule that function as signals. When it binds, it causes inhibition of transcription by causing the repressor to bind to the DNA and block transcription.

Lac operon info

On main chromosome, looks like this

Readthrough

If you transcribe through a terminator, this is what it is called

Rho

Has helicase actvity in bacterial termination of transcription. Utilizes ATP, which makes it have ATPase activity, to “yank” RNA off the DNA template and cause RNA polymerase to dissociate due to change in conformation.

HATs/HDACs

Histone acetyl transferase (loosens chromatin by adding acetyl group to histone protein to remove charge off of arginine and lysine positive charged amino acids)/ Histone deacetylases (tightens chromatin by taking acetyl group off and increasing the positive charge on histone). This is a eukaryotic thing.

CAP site

Called catabolite activator protein. Binds CBP to drive transcription

Promoter, operator, and CAP site are all…

Molecular switches

CBP

CAP binding protein. Binds to CAP to drive transcription. Needs cAMP (cyclic AMP) to be able to bind to the CAP site.

Is cAMP a nucleotide

Ya

Bacteria prefer which one: glucose or lactose

Glucose

If glucose levels are high, cAMP is…

Low/not present

If glucose levels are low, cAMP is…

High

Diayxic growth

Bacteria showing preference in sugar source

LacZ gene codes

B (beta) galactosidase. This enzyme splits lactose into glucose and galactose and has a side reaction to turn lactose into allolactose (slight structural change).

Inducer for lac operon

Allolactose

Lac Y gene codes for…

Permease (protein in cell wall involved in transport: moves lactose extracellularly into the cell)

Lac A codes for…

Transacetylase (covalently modifies lactose and analogues and it is not clear what its functional necessity is)

Lac I

On all the time(constituitive), it is a regulatory gene that is NOT part of the lac operon. However, it codes for the repressor that binds to the operator as a dimer to physically block RNAP from binding to the promoter and initiating transcription.

Lac repressor

If allolactose binds, then the repressor finds off and transcription of the lac operon can occur. Need low glucose and high lactose for transcription.

Fertility factor

Merozygotes/partial diploids

When only some of the chromosome of the other bacteria is brought into another bacteria so they have 2 copies of some genes but not all.

Hfr strain

High frequency recombination strain. This is when the F factor recombines into the bacterial main chromosome. The F factor can then excise itself, but sometimes it doesn’t do it well and the F factor can take some of the other genes from the main chromosome with it to create merozygotes.

Jacob and Morod 1950’s hypothesis

Either lac I codes for a repressor that when broken is off all the time or an activator that when broken is on all the time.

Symbols in nomenclature for lac operon

+ means wild type (wt), - means nonfunctional (think of it as the protein or whatever it is isn’t made), / signals that the that the second gene is gonna be shown, O^c means constitutive mutation so that the operon is always on and repressor cannot bind, I^s means super repressor, so that when this repressor is made it never comes off.

IPTG

Allolactose analogue that has more stability which is why we use it. Its synthetic.

can genes ever truly be turned off? What is this in the case of the lac operon?

Nope. In the case of the lac operon, some of the pumps of the lactose permease is always there, so then some of the lactose extracellularly can enter the cell and actually start the lac operon process.

How did they find out if Lac i was an activator or repressor?

They used merozygote that has in one gene mutation I^- and the rest is wild type and the other gene has all wild type. They used a control that was haploid and its only mutation was the I^-. They used these conditions: no glucose and no inducer, and no glucose with presence of inducer. They used a beta-ONPG to tell if beta galactosidase was made because that protein will turn the originally clear B-ONPG and turn it yellow. They used spectrophotometer to quantitate how much yellow is actually there. The results: the haploid control strain got 100% beta galactosidase with and without the inducer. The merozygote under no inducer had 100% expression of the lac Z gene, and with inducer had 200% expression. This showed that it was a repressor. If it was an activator, both under induction and without induction it would have been 200%.