Chapter 16

What are the two loops, or structures that the Trp operon can form, to indirectly assess Trp concentration?

- 1+2, 3+4 Attenuation (terminates transcription) - 2+3 Antitermination

What is the structure of the Trp operon? What does this structure have a lot of power over?

- Has long regulatory region, with a start codon for regulatory region, and 2 Trp codons in region 1 - It also has a Trp E gene, with its own start codon - This has a lot of power for regulating translation.

Explain how the antitermination loop forms in the Trp operon when tryptophan is low, during transcription/translation.

- If Trp is low, the ribosome will stall over the Trp codons. - This is due to the fact that Trp codons are entering a cell, with few tRNA's charged with Trp, so there would be few charged tRNA's that could deliver the Trp to the A site. - So the Ribosome will stall because Ribosome's stay moving from the delivery of AA's to A site via tRNA's that correctly match the codon-anticodon situation at A site. - The stalling of the ribosome causes the formation of the anti-termination loop, meaning transcription wont terminate and the ribosome will continue to transcribe trp.

What will be the most likely effect on transcription of the trp structural genes if a mutation that changes the tryptophan codons in region 1 of the mRNA 5' UTR into codons for alanine occurs?

- In the wild-type trp operon, low levels of tryptophan result in the ribosome pausing in region 1 of the mRNA 5' UTR. The pause permits regions 2 and 3 of the mRNA 5' UTR to form the antiterminator hairpin, allowing transcription of the structural genes to continue. - If alanine codons have replaced tryptophan codons, then under conditions of high alanine, the stalling of the ribosome will not occur. The attenuator will form, stopping transcription. The ribosome will stall when alanine is low, so transcription of the structural genes will occur only when alanine is low

What happens to the lac operon when lactose is present?

- Lactose will bind to the regulatory protein, inactivating it - This will cause it to be unable to bind to the lacO operator, or if it was bound it will fall off - This enables RNA polymerase to bind, allowing for the transcription and translation of permease, which will turn lactose to glucose and galactose.

In an inducible system what happens when an inducer is present? (positive inducible system)

- The inducer binds to the repressor and causes the repressor to fall off. - The repressor or regulatory protein has two shapes, a DNA binding site, and an inducer binding site. when an inducer is bound it changes the shape of the DNA site and is lost, it falls off of the operator and the RNA polymerase can bind and transcription and translation can occur.

What happens to cAMP when glucose is high?

- When glucose is high, there is low cAMP. - There is little cAMP binded with CAP, so we don't get binding there, so RNA polymerase is not recruited so there is little to no translation/transcription - NOTE, the promoter is open and the operator is not blocked so once in a while RNA polymerase will initiate transcription.

What is the role of cAMP in the lac operon when glucose is low

- When glucose is low, the concentration of cAMP will increase. - cAMP will bind to CAP, and they will then bind to the cAMP binding site upstream of promotor. - This will allow for the recruitment of RNA polymerase which will then allow lactose to be made into glucose

Explain the feedback mechanism of the Trp operon when tryptophan is low, and when tryptophan is high.

- When tryptophan is low, there is little to none available to bind to the tryptophan binding site of the regulator protein. This will cause the repressor to be unable to bind to the operator, allowing the RNA polymerase to bind, initiate transcription and translation, making tryptophan - BUT now than Tryptophan levels are high, the tryptophan will bind to the repressor activating it. It will not bind to the operator, inhibiting the making of tryptophan

What is the relationship between cAMP, CAP, and DNA?

- cAMP has alpha helices that connect with major groove area, bending the DNA - cAMP in the CPA protein is what causes the CAP to change shape making it able to bind. - In the absence of cAMP, CAP cant bind because the distance between the alpha helices changes and gets larger in the absence of cAMP, so the protein (CAP) does not fit

What does the lac operon do? (use E. coli as an example)

-Allows the cell to use lactose when glucose is not present. -E.coli will preferentially use glucose as a source of energy. - If it is not present a permease allows lactose to be brought into the cell then beta-Galactosidase, turns lactose to allolactose, and then allolactose can be processed into galactose and glucose which the cell can use as an energy source

What is the relationship between an operon and a regulator protein?

-An operon will always be paired with a regulatory protein. -The regulatory protein will have its own promotor, so it is not physically associated with the operon. - The regulatory protein is constitutively expressed, meaning it is always being made in order to regulate the operon.

What are the levels of gene control in eukaryotes? (5 levels)

-Compact DNA through alteration of structure becomes relaxed DNA - Relaxed DNA through transcription becomes pre-mRNA - Pre mRNA through mRNA processing becomes processed mRNA - Processed mRNA we can still have some control over due to poly A tail and RNA stability. Processed RNA through RNA stability translation we can make inactive proteins - Inactive proteins through posttranslational modification can become an activated protein

Explain the process of the repressor binding to lacO and why it stops transcription of structural genes. (when no lactose is present)

-The active regulator protein (repressor) is made from regulator gene (lacI). - It binds to the lacO. - LacO overlaps the promoter, so when repressor binds to lacO it also blocks the promotor which is why transcription of structural genes is stopped.

What is the function of allolactose in regulation of the lac operon? A. None of the options is correct B. Promoter C. Activator D. Regulatory protein E. Repressor

A

What is a riboswitch?

An mRNA that can regulate its own transcription and translation (gene expression). By having sequences of RNA on mRNA that form secondary structures upstream or including the ribosomal binding site.

Why do binding proteins only interact at the major groove?

Because that is where we get sequence specific interaction. We don't get sequence specific interactions in minor grooves.

Proteins with DNA binding motifs predominantly bind to the of DNA_________ in a sequence specific manner. A. Minor grooves B. Deoxyribose sugar C. Major grooves D. Phosphate groups E. Paired nitrogenous bases

C

The tryptophan riboswitch is critical for proper transcriptional and translational regulation. The formation of 1+2 and 3+4 secondary structures of 5′ UTR region mRNA from the trp operon is triggered when A. The structural gene transcription within the trp operon gets initiated. B. The repressor protein fails to bind to the operator. C. The tryptophan level inside the bacterial cell is high. D. The tryptophan level inside the bacterial cell is extremely low. E. There is a spontaneous mutation introduced into the 5′ UTR.

C

Under which of the following conditions would a lac operon produce the greatest amount of beta-galactosidase? Explain Lactose present: Glucose Present: Condition 1: Yes No Condition 2: No Yes Condition 3: Yes Yes Condition 4: No No

Condition 1 will result in the production of the maximum amount of the beta-galactosidase. - For maximum transcription, the presence of lactose and the absence of glucose are required. Lactose ( or allolactose) binds to the lac repressor reducing the affinity of the lac repressor to the operator. This decreased affinity results in the promoter being accessible to RNA polymerase. - The lack of glucose allows for the increased synthesis of cAMP, which can complex with CAP. The formation of the CAO-cAMP complex improves the efficiency of the RNA polymerase binding to the promoter, which results in the higher levels of transcription from the lac operon.

Under which of the following conditions would a lac operon produce the least amount of beta-galactosidase? Explain Lactose present: Glucose Present: Condition 1: Yes No Condition 2: No Yes Condition 3: Yes Yes Condition 4: No No

Condition 2 will result in the production of the least amount of beta- galactosidase. - With no lactose present, the lac repressor is active and binds to the operator, inhibiting transcription. -The presence of glucose results in a decrease of cAMP levels. A CAP-cAMP complex does not form, and the RNA polymerase will not be stimulated to transcribe the lac operon.

Which of the following generally get transcribed constitutively? A. Operon B. Operator element C. Structural gene D. Regulatory gene E. Promoter element

D

What will be the most likely effect on transcription of the trp structural genes if deletions in region 4 of the mRNA 5' UTR occur?

Deletions in region 4 will prevent formation of the attenuator by the 5' UTR mRNA. Transcription will proceed

Which of the following facts about riboswitches is CORRECT? A. The secondary structure that forms riboswitches typically contains a base stem and several branching hairpins. B. Binding of certain molecules to the riboswitches results in the formation of specific secondary structures of mRNA. C. Certain molecules that bind to riboswitches may act as repressors or inducers of transcription. D. Riboswitches are found in bacterial cells, archaeal, fungal, or plant cells. E. All are correct

E

True or False When lactose is low, and the lac operon operator is bound by a lac repressor, transcription/translation can still take place.

False. The lac repressor would stop the promotor from undergoing transcription/translation of the structural genes. This is due to the fact that lactose is low, and so the repressor is telling the cell not to make permease because the cell does not have enough substrate.

What will be the most likely effect on transcription of the trp structural genes if deletion of the string of adenine nucleotides that follows region 4 in the 5' UTR occurs?

For the attenuator hairpin to function as a terminator, the presence of a string of uracil nucleotides following region 4 in the mRNA 5' UTR is required. The deletion of the string of adenine nucleotides in the DNA will result in no string of uracil nucleotides following region 4 of the mRNA 5' UTR. No termination will occur, and transcription will proceed.

What is the function of the zinc finger binding proteins?

Have zinc ions, and alpha helix that will fit into regions of the major groove, usually that are not too far from each other.

What is the position of the ribosome when region 3 is transcribed in both high and low Trp levels?

High: ribosome covers region two Low: covers region one

What will be the most likely effect on transcription of the trp structural genes if a mutation that creates a stop codon early in region 1 of the mRNA 5' UTR occurs?

If region 1 of the mRNA 5' UTR is free to pair with region 2, then regions 3 and 4 of the mRNA 5' UTR can form the attenuator. An early stop codon will result in the ribosome "falling off" region 1, allowing it to form a hairpin structure with region 2. Transcription will not occur because regions 3 and 4 are now free to form the attenuator.

What will be the most likely effect on transcription of the trp structural genes if deletions in region 2 of the mRNA 5' UTR occur?

If region 2 of the mRNA 5' UTR is deleted, the antiterminator cannot be formed. The attenuator will form and transcription will not occur.

Describe the effect on the ribosome binding site when no regulatory protein is present. (riboswitch)

If regulatory protein is absent, the ribosomal binding site wont close, and is available and translation will happen. (the gene made is the regulatory protein)

What will be the most likely effect on transcription of the trp structural genes if a mutation that prevents the binding of the ribosome to the 5' end of the mRNA 5' UTR occurs?

If the ribosome does not bind to the 5' end of the mRNA , then region 1 of the mRNA 5' UTR will be free to pair with region 2, thus preventing region 2 from pairing with region 3 of the mRNA 5' UTR. Region 3 will be free to pair with region 4, forming the attenuator or termination hairpin. Transcription of the trp structural genes will be terminated. Essentially, no gene expression will occur.

What is the significance of the lac operon?

It was the first operon to be understood, we learned about the operon model from this.

What is the idea of the operon model?

One regulatory region, and a variety of structural genes that are, transcribed at the same time from one regulatory region

What is the meaning to the letter is the lac operon? P- not part of lac operon but part of system I- not part of lac operon but the system P O Z Y A

P= promotor for gene 1 (regulator protein; repressor) I= gene product (of gene 1) that binds the operator P= promotor for Z, Y and A O= operator= repressor binding site Z, Y, and A= structural genes, codes for permease and 2 galactosidases

What is a negative repressible operon?

The Inactive regulator repressor protein cant bind to the operon to stop the system until it is turned on. It needs a corepressor to bind to the inactive regulator, unlocking the DNA binding site so it can inactivate the pathway. - Common in feedback systems (corepressor product tells enzyme to stop making product_

What is the relationship between an operon and structural genes? Think of biochemical pathways.

The genes and the operon are involved in a particular pathway such that we can take a precursor, have some intermediates, and eventually get the product you are looking for through biochemical pathways.

What does the operon consist of

The operon consists of the promoter (binded to operator) } regulatory region, and structural genes.

What will be the most likely effect on transcription of the trp structural genes if deletions in region 3 of the mRNA 5' UTR occur?

The trp operon mRNA 5' UTR will be unable to form the attenuator if region 3 contains a deletion. Attenuation or termination of transcription will not occur, resulting in continued transcription of the trp structural genes.

What is the function of the DNA binding protein Helix-turn-helix?

The turn in the protein gives the flexibility so that we can perfectly match the alpha helix into the major groove. - A dimer (2 of them) will help to bind to DNA and activate transcription

What is the function of the leucine zipper binding protein?

Usually have 2 alpha helices connected by leucine residues. Will shove themselves into DNA and bind regularly.

Describe the effect on the ribosomal binding site when a regulatory protein is present. (riboswitch)

When there is a regulatory protein is bound in the riboswitch upstream of ribosomal binding site. The riboswitch, and the ribosomal binding site becomes double stranded, and closes, thus we wont have translation.

Explain how the terminator loop forms in the Trp operon, when tryptophan levels are high.

When tryptophan levels are high the ribosome does not stall at the Trp codons. This causes the terminator loop to form and mimics Rho- independent termination. The loop forms with such force that it kicks off the RNA polymerase ending transcription

A mutant strain of E. coli produces β-galactosidase in the presence and in the absence of lactose. Where in the operon might the mutation in this strain occur?

Within the operon, the operator region is the most probable location of the mutation. - If the mutation prevents the lac repressor protein from binding to the operator, then transcription of the lac structural genes will not be inhibited. Expression will be constitutive. Outside of the operon, a mutation in the lacI gene that inactivates the repressor or keeps it from binding to the operator could also lead to constitutive expression of the structural genes.

What is a negative inducible operon?

You have a protein bound. When the regulatory protein is bound the system is off (negative part). We have to have something happen that is going to get that protein to fall off, and then we can accurately initiate transcription (inducible part)