Wright 6 - Prokaryotic Gene Regulation

Question 1

What is a gene?

Answer 1

A stretch of DNA that is transcribed into RNA. It includes the regulatory elements that control its transcription.

Question 2

How can the sequence of a bacterial promotor be identified?

Answer 2

1. Add RNA polymerase and DNA fragment to a test tube allowing protein-DNA binding. (this protects the DNA that the protein is bound to)
2. Add to test tube DNase I to digest unprotected DNA
   3, Purify the protected DNA, clone and sequence

Question 3

How can analyzing mutant with enhanced or diminished rates of transcription help identify the sequence of a bacterial promotor?

Answer 3

Mutations that change the nucleotide sequence of a bacterial promotor from the consensus sequence diminish the rate of transcriptional initiation.

Question 4

What are the threes stages of RNA synthesis?

Answer 4

1. Initiation (RNA polymerase recognizes a promotor, melts the TATA box and initiates the incorporation of the first ribonucleoside triphosphate into the nascent RNA)
2. Elongation (growth of the RNA polymer in the 5’ to 3’ direction)
3. Termination (synthesis of the RNA polymer stops at precisely defined sites coded by the DNA template)

Question 5

What subunit of RNA polymerase must bind to it to control the initial binding of RNA polymerase to the promotor?

Answer 5

The sigma factor subunit

Question 6

What does the omega subunit of RNA polymerase do?

Answer 6

It is not essential for transcription, but it helps stabilize the RNA polymerase

Question 7

What are the subunits of the core RNA polymerase? Of the RNA polymerase holoenzyme?

Answer 7

• 2 alpha
• 1 beta prime
• 1 beta
• 1 omega

The sigma factor binds to create the RNA polymerase holoenzyme, which is required for promotor recognition and initiation of transcription. The sigma factor is release from the holoenzyme after initiation to participate in the recognition of other promotors. The core enzyme proceeds to complete elongation and termination of the RNA transcript

Question 8

What is the irreversible step in initiation of transcription translation?

Answer 8

The opening of the promotor complex with the sigma factor and RNA polymerase holoenzyme

Question 9

When does protein synthesis require energy? (two steps)

In what form does that energy come?

Answer 9

• The charging of tRNA with amino acid binding (ATP hydrolysis)
• Loading tRNA bonds to ribosome by transferring amino acids to growing polypeptide (GTP hydrolysis)

Question 10

When biosynthetic genes in a prokaryote (such as E. coli.) are clustered together and under the control of a single promotor, is there one start site next to the promotor? Or a start site at each biosynthetic gene?

Answer 10

Trick question.

There is one start site for mRNA transcription, but a start site for protein synthesis at EACH gene. Each translation start site needs a ribosome, this creates a polyribosome on the mRNA.

Question 11

Who deciphered the regulation of the lactose operon?

Answer 11

Francois Jacob and Jacques Monod

Question 12

How is lactose metabolized?

Answer 12

When it is transported from growth medium into the cell with permease proteins, it then undergoes breakdown by beta-galactosidase to two molecules of glucose.

Question 13

What happens when lactose is added to growth medium with E. coli.?

Answer 13

Causes induction of beta-galactosidase synthesis in E. coli. More lactose metabolizing enzyme is produced over time until it is 6% of all the protein made by a cell. The question is, how does this induction happen? It is a sort of gene regulation.

Question 14

What happens when there are low levels of beta-galactosidase in a cell inundated with lactose?

Answer 14

Beta-galactosidase convertes lactose to allolactose (an isomer), this acts as a functional ligand of the lactose repressor

Question 15

How does allolactose block transcription of the lac operon (lactose operon).

Answer 15

1. Allolactose binds to the repressor protein and activates it by a conformational change.
2. The active repressor protein binds to DNA at the operator part of the lac operon, this block RNA polymerase to that no RNA is made.

Only lacl mRNA is made (from the regulatory gene), this is translated into the repressor protein

Question 16

What is the conformational transition that the lactose repressor undergoes upon binding with allolactose? Is this reversible?

Answer 16

The DNA binding domain is masked when allolactose binds. This causes the repressor protein to become inactive, and beta-galactosidase is produced by the cell. When there are high levels of lactose, allolactose is not made by beta-galactosidase and the repressor remains active with an open DNA binding domain, inhibiting production of beta-galactosidase, lactose permease and transacetylase.

Question 17

How do high levels of lactose inhibit the repressor protein at the lac operon and induce more beta-galactosidase to be made?

Answer 17

1. Lactose is converted to allolactose by beta-galactosidase
2. Allolactose binds to the repressor protein made by the lacl regulatory gene of the lac operon
3. The repressor protein becomes inactive and allows RNA polymerase to transcribe RNA for beta-galactosidase, permease and transacetylase unhindered