Genetics module 12

4.What is a homopolymeric protein?

"homodimer" that contains two identical polypeptides in the functional protein which binds with one of the inverted repeat segments

16.How does the cellular concentration of tryptophan affect whether the attenuator region of the trp operon forms an anti-termination stem-loop or a termination stem-loop? What codons are critical to this process?

1) The polypeptide-coding sequence overlaps the entirety of leader region 1, and the stop codon is immediately adjacent to region 2; (2) codons 10 and 11 of the mRNA specify tryptophan, making completion of translation dependent on tryptophan availability; and (3) region 4 is followed immediately by a poly-U string, a feature associated with intrinsic termination of transcription.

attenuation

A gene regulatory mechanism that fine-tunes transcription to match the momentary requirements of the cell, achieving a more or less steady state of compound availability.

repressor

A transcription factor that binds to regulatory sequences associated with a gene and represses that gene's expression.

12.In the chart below (from Jacob et al. 1961), data is shown for measurements of the ß-galactosidase protein and total bacterial proteins in a bacterial cell culture. a.What is the probable inducer added to the cell culture? b.What impact does the inducer have on ß-galactosidase production? c.What is the inducer doing at the molecular level to have this effect? d.Why do you think total bacterial protein also increasing throughout this experiment?

A. inducer is allolactase B. Allolactase concentration allows for RNA polymerase transcription of lacz to produce beta-galactosidase C. allolactase binds to repressor protein to inhibit its blockage of RNA polymerase] D. Transcription to translation is increasing protein production

8.What is the role of cyclic AMP (cAMP) in regulation of the lac operon?

AMP is a "hunger signal" made by E. coli when glucose levels are low. cAMP binds to CAP, changing its shape and making it able to bind DNA and promote transcription

antisense RNA

An RNA molecule that is complementary to a portion of a specific mRNA.

inducer

An accessory molecule that binds to a protein that leads to activation of gene expression. The inducer can bind to a repressor protein and prevent its function or bind to an activator protein and stimulate its function.

partial diploid

An exconjugant bacterium that acquires a second copy of one or more genes by conjugation with an F'F′ donor cell.

anabolic pathway

Anabolic pathways are those that require energy to synthesize larger molecules.

catabolic pathway

Catabolic pathways are those that generate energy by breaking down larger molecules.

1.Give one example of a constitutively expressed gene and one example of a gene with regulated expression - any organism is fine.

Constitutively expressed gene - ribosomal gene Regulated expressed gene - bacterial gene

3.What are the two binding domains of repressor and activator proteins? To which of these sites do co-repressors, inhibitors, inducers and activators typically bind?

DNA- binding domain - is responsible for locating and binding operator DNA sequence or other target regulatory sequences Allosteric domain - binds a molecule or protein which causes a change in conformation of the DNA-binding site. activator protein - effector binds to the activator binding site and causes transcription inhibitors bind to and prevent activator function Repressor protein - inducer binds to cease repression corepressor binds to cause repression

6.Describe E. coli transcriptional response in the lac operon to each of the following scenarios: glucose present/lactose absent, glucose present/lactose present, glucose absent/lactose present, glucose absent/lactose absent.

E. coli produces three polypeptides needed to metabolize the carbohydrate lactose as an energy source glucose present/lactose absent - glucose is present to provide energy there is no alloctaose to bind repressor. there is no cap-camp complex to bind cap site glucose present/lactose present - glucose is present to provide energy, absence of camp prevents positive transcription regulation, but allolactose is present and acts as an inducer to allow a small amount of transcription glucose absent/lactose present - cap-camp forms, but no allocatose present to block repressor binding at operator glucose absent/lactose absent - inducer and cap-camp are available to induce and positively regulate transcription

direct repeat

Identical or nearly identical DNA sequences in the same orientation that are separated by intervening DNA.

inverted repeat

Identical or nearly identical DNA sequences located on the same molecule but with opposite orientations.

2.Contrast negative and positive control of transcription - what type of regulatory proteins does each use, and what is the impact on transcription?

Negative control - condition where binding of a repressor protein to a regulatory DNA sequence with consequence of preventing transcription of a gene or a cluster of genes. repressor proteins - prevents gene transcription positive control - binding of an activator protein to regulatory DNA, with the result of initiating gene transcription. activator protein - initiates or increases gene transcription

stem-loop RNA

Stem-loop is an essential unit of the structure of single-stranded RNA or DNA. A stem-loop consists of a stem, double helix, and a loop that links the stem. The length of the loop is typically 3-8. Tetraloops, stem-loops with four nucleotides in the loop, are frequently found in RNA.

14.What is the role of tryptophan in regulation of the trp operon, and how does this qualify as 'feedback inhibition'?'

acts as corepressor major control mechanism for regulating trp operon is the presence/absence of tryptophan

11.Which molecule acts as an inducer for the lac operon? Where does this molecule come from?

allolactase comes from lactose converted by beta-galactosidase

allosteric interaction

allostery - property of some enzymes to change conformation at the active site as a result of a binding substance at a different site

7.What is the role of the operator region in the lac operon? Where does the operator reside in the operon, and why is this location important for regulating transcription?

binds repressor protein to block transcription operator is located in the regulatory region of the operon location is the regulatory region where: § promoter (lacP)- binds RNA polymerase § operator (lacO) - binds lac repressor protein § CAP-cAMP region - binds activator protein

operon

cluster of genes undergoing coordinated transcriptional regulation by a shared regulatory region

regulated expression

condition in which gene expression is controlled at the transcriptional level in response to changing environmental conditions

constitutive expression

continuously expressed under normal cellular conditions

5.What is the common structural motif among DNA-binding regulatory proteins in bacteria?

helix-turn-helix (HTH) A DNA-binding protein domain consisting of 2 alpha helices: one helix binds to a specific DNA sequence, and the second helix stabilizes the interaction

9.Describe how the lac operon is an example of both negative and positive control of transcription.

lac operon has conditions that need to be met to either initiate or suppress transcription

10.What gene codes for the lac repressor protein? Where is this gene located?

lacO regulatory region

15.How many inverted repeat sequences reside in the attenuator region of the leader region in the trp operon? How do these form stem-loop structures?

mechanism involves four inverted repeat sequences residing in attenuator region of leader region (trpL) § 1-2 stem loop - 'pause stem loop' § 2-3 stem loop - ' anti-termination stem loop' § 3-4 stem loop - 'termination stem loop

13.Describe the structure of the leader region of the trp operon? Where does this region reside in the trp operon?

§ 'leader' region § leader region contains an attenuator region with transcription termination site after trpP (trp promoter)

18.Describe two significant mutations that could occur to genes coding for repressor proteins, and their resulting effect on transcription.

§ a number of base substitution mutations to the operator region of the lac operon result in constitutive expression § generally, these mutations disrupt the symmetry of the inverted repeat sequences present in the operator region § prevents lac repressor binding § mutations to the trpL region of the trp operon can also result in constitutive expression § substitution mutations to regions 3 and 4 may inhibit 3-4 stemloop formation § intrinsic termination avoided

17.Why would significant mutation to a promoter or operator region result in a constitutive effect on an operon?

§ operator-site mutations may prevent repressor protein binding ('on') § repressor protein DNA-binding site mutations may prevent repressor binding ('on') § repressor protein allosteric-site mutations may prevent inducer binding ('off')

lac operon structure

§ regulatory region § promoter (lacP)- binds RNA polymerase § operator (lacO) - binds lac repressor protein § CAP-cAMP region - binds activator protein § Three structural genes § lacZ - encodes β-galactosidase § lacY - encodes permease § lacA - encodes transacetylase

.19.What key region of the mRNA molecule is impacted in translation regulation in bacteria (for example by repressor protein binding

§ translation repressor proteins bind mRNA near Shine-Dalgarno sequence § interferes with ribosomal binding of mRNA § small RNAs (sRNA) can activate or repress RBS-ribosome interaction