Messengers, TCS, and Chemotaxis - Shanks

Question 1

How do bacteria generally respond to their environment?

Answer 1

They alter transcription and RNA/protein modifications.

Question 2

What do sigma factors do?

Answer 2

Sigma factors associated with RNAP recognize the -10 to -35 promoter sequence where RNAP binds. Sigma factors direct the specificity of transcription and subsequent gene expression.

Question 3

How are proteins (transcription factors) important to gene regulation?

Answer 3

Tfs can either be positive or negative regulators that affect the binding of polymerase and the specificity of where it binds.

Question 4

What does the variety of sigma factors present in a species of bacteria say about it?

Answer 4

Organisms with a more diverse range of sigma factors can deal with more diverse environments by having more ways to alter gene expression.

Question 5

What regulates sigma factors?

Answer 5

1. Anti sigma factors can bind to sigma factors which shuts down transcription of certain genes associated with that sigma factor.
2. There are also transcription factors which can direct expression of certain sigma factors.
3. Varying stabilities in certain cellular environments.

Question 6

What are 3 ways transcription factors repress transcription?

Answer 6

1. Steric hinderance blocking RNAP
2. Form DNA loops blocking RNAP
3. Can bind to positive regulators preventing recruitment of RNAP

Question 7

What are ways transcription factors activate transcription?

Answer 7

1. Bind to DNA and attract or stabilize RNAP interactions.
2. Can change structure of DNA to enhance binding

Question 8

What is a co-inducer?

Answer 8

Molecules that bind to proteins and modulate their function.

Question 9

What is the general structure of a two-component system?

Answer 9

A histidine-kinase with a highly variable sensor domain which transduces a signal via phosphorylation cascades to a response regulator which acts as a transcription factor.

Question 10

What is an operon?

Answer 10

Multiple genes regulated by a single promoter. Genes on an operon are transcribed as a single RNA transcript, but translated individually.

Question 11

Why are operons useful to bacteria?

Answer 11

Operons usually control multiple genes needed for a single process. This allows for rapid response since all the proteins needed are transcribed and translated at once.

Question 12

How do operons contribute to horizontal gene transfer?

Answer 12

Since an operon codes for whole functional units, bacteria picking up plasmids that contain operons are much more likely to be able to utilize the machinery they code for.

Question 13

Describe regulation of the lac operon

Answer 13

In the absence of lactose, the lac repressor is bound to the DNA preventing transcription. When lactose and glucose are both present, it acts as a co-inducer, changing the shape of the repressor so that it is no longer bound to DNA. RNAP can then bind and transcribe the lac operon genes at a basal level. Additionally, when only lactose is present, cAMP (a positive txn factor) binds to CAP and upregulate expression of the lac operon.

Question 14

Describe Polar Effects

Answer 14

Mutations in a gene (especially in an operon), can have effects on neighboring genes. For example, if an upstream gene in an operon is mutated in such a way that the transcribed mRNA is unstable, there will be no expression of downstream genes. This is why you must do complement experiments when studying gene function in prokaryotes.

Question 15

Can genes have multiple promoters?

Answer 15

Yes, for example sarA

Question 16

How do miRNAs impact gene expression.

Answer 16

miRNAs can have a negative impact by blocking translation, destabilizing mRNAs or have a positive impact by facilitating translation. So, there is regulation at the transcriptional and post-transcriptional level.

Question 17

Describe secondary messengers

Answer 17

Secondary messengers amplify environmental stimuli which can lead to modulation of transcription. For example, in low glucose environments, adenylate cyclase converts ATP to cAMP which binds to CRP (txn factor).

Question 18

What is a reporter fusion?

Answer 18

You can measure promoter activity (transcription) or protein production (translation) by utilizing a reporter gene such as GFP or luciferase.

Question 19

Describe Northern Blotting

Answer 19

Extract RNA from a sample and run it on a gel. The gel is transferred to a membrane and radioactively labeled to visualize on x-ray film. Used to measure amount and size of transcripts.

Question 20

Why do you need a no RT control when using RT-qPCR to measure txn?

Answer 20

You need to make sure your primers aren’t amplifying genomic DNA since cDNA of mRNA transcripts are the same sequence as the gDNA. You can use DNASE to remove gDNA.

Question 21

What is the purpose of a housekeeping gene in RT-qPCR?

Answer 21

To compare levels of expression of the target gene to a gene that is expressed normally under the same conditions.

Question 22

Describe direct and indirect regulation and how you can test for it.

Answer 22

Txn factors can either directly influence expression by directly interacting with DNA, or they can influence expression by interacting with another protein that binds to DNA (indirect). You can test it with EMSA, DNase footprinting, or ChIP seq.

Question 23

Describe EMSA

Answer 23

You label your DNA sequence of interest. You incubate the sample with purified protein and another without. If txn factor of interest is bound, it will migrate slowly through gel.

Question 24

What does DNase footprinting tell you?

Answer 24

Where your txn factor is binding to your DNA

Question 25

Describe Steps in ChIP

Answer 25

1. Cross-link DNA interacting with proteins.
2. Shear DNA-protein complex
3. Immunoprecipitate with antibody specific to protein that is bound to DNA
4. Remove cross-links, then do PCR or NGS

Control would be to use an antibody not specific to the protein.

Question 26

What is chemotaxis?

Answer 26

Flagella responding to chemical signals by 2-component regulators to move towards or away from certain substrates. More random tumbling when moving away from something.