Exam 3 Lecture 22

Question 1

Where is the attenuator region of the Trp operon?

Answer 1

At the end of the regulatory region in between the operator and the first Trp gene.

Question 2

What does the attenuator in Trp do?

Answer 2

Adds an extra level of regulation to fine-tune down-regulation depending on which regions of it bind together. Functions on the newly transcribed mRNA.

Question 3

What 10 parts make up the Trp operon and what order are they in?

Answer 3

1. Promoter for TrpR
2. trpR the regulatory gene for the aporepressor
3. Promoter for the main gene sequence
4. Operator Region
5. Leader Region trpL- attenuator is in the middle of it
6. trpE gene
7. trpD gene
8. trpC gene
9. trpB gene
10. trpA gene

Question 4

How does an attenuator work?

Answer 4

Translation of the trp gene starts and the attention sequence is made first. A ribosome can hop on and get ready to start transcription but the first attenuation sequence of the mRNA has 4 regions that can bind to each other form stem-loop complexes. Depending on what regions bind with what the response is different.

Question 5

What are the 3 responses of the attenuation of the trp operon?

Answer 5

1. Pause to allow ribosome time to start translation
2. Antiterminator loop pauses ribosome/translation allows transcription
3. Termination

Question 6

What happens when attenuator regions 1 and 2 bind

Answer 6

Pause loop forms

Question 7

What happens when trp attenuator regions 2 and 3 bind?

Answer 7

Anti-terminator loop

Question 8

What happens when trp atentuator regions 3 and 4 bind?

Answer 8

Terminator loop is formed

Question 9

Which trp attenuator loop forms first?

Answer 9

Regions 1 and 2 to pause transcription till the ribosome gets on causes the loop to release so transcription can continue.

Question 10

When does trp attenuator regions 2 and 3 to bind and why?

Answer 10

In times of low trp when there aren’t enough tRNA with trp on them, the ribosome will stall at an area of T’s on the mRNA in region one. There isn’t a tRNA-T to keep going! then regions 2 and 3 will bind since it is more energetically favorable than the 3 and 4 binding.

2 and 3 is the anti-terminator loop and it allows the mRNA transcription to continue and transcribe trpE but stops translation.

Question 11

What happens in high levels of trp to the attenuator region if for some reason high levels of trp didn’t already bind to the aporepressor to make a holorepressor and bind to the operator to stop transcription?

Answer 11

Ribosome hops on translates through the trp area in region 1 gets to region 2 and while covering region 2 gets to a stop codon allowing regions 3 and 4 to bind- the termination loop.

Question 12

How does the trp termination attenuator loop work?

Answer 12

The termination loop is G-C rich followed by a bunch of UUUU acts and an intrinsic transcription stop. It also binds to polymerase causing it to release before reaching trpE and no trp mRNA to be made.

Question 13

3 main types of regulatory RNAs?

Answer 13

1. Riboswitches
2. asRNA (antisense made from the antisense DNA strand while the sense mRNA is being made)
3. sRNA (small) encoded in regions between genes

Question 14

What do regulatory RNAs do?

Answer 14

Regulate mRNA translation sometimes transcription by

• interacting with mRNA
• and proteins

Question 15

What does a riboswitch do?

Answer 15

Riboswitches control transcription or translation via stem-loop and/or ligand binding.

Usually at the 5’ UTR

Think trp attenuator

Question 16

Example of a riboswitch?

Answer 16

The trp attenuator is a riboswitch

Question 17

asRNA are how long?

Answer 17

700-3000 nt long

Question 18

Where are asRNA ‘s made from?

Answer 18

The antisense DNA strand while the sense mRNA is being transcribed.

Regulate only that gene or operon

Question 19

Two main ways asRNA works?

Answer 19

1. RNA polymerase will get on antisense strand and collide with sense RNA polymerase
2. The antisense strand is transcribed without a collision and goes on to bind to the sense mRNA and interfere during either transcription or translation.

Question 20

How does asRNA work to interfere with transcription (other than polymerase collision)?

Answer 20

Binds tightly to mRNA to being made and can stop transcription like an attenuator does or an intrinsic transcription stop.

Happens on plasmids often.

Question 21

Two other ways asRNAs work?

Answer 21

1. Endonuclease activity either blocking or tagging it for destruction
2. Translational blocking either directly or indirectly

Question 22

Specific example of asRNA?

Answer 22

The S-box or SAM (s-adenosylmethionine) riboswitch

Antisense RNA and a riboswitch both at the same time!!

Also uses transcription interference RNA poly collision!

All three concepts in one!

Question 23

How does the SAM/S-box asRNA work?

Answer 23

RNA poly get on antisense DNA of ubiG-mccB-mccA operon

The first bit of the asRNA codes the switch

When not bound to anything aka low levels of ligand SAM anti-terminator loops are made and transcription continues causing and RNA polymerase collision with sense strand RNA poly and termination of transcription.

BUT when SAM is around it binds and causes a termination loop allowing sense RNA strand to be made.

Question 24

What are sRNA’s? How long?

Answer 24

Small RNAs

100-200 nts

Question 25

What do sRNAs do?

Answer 25

Bind to mRNA and regulate translation up or down as an extra layer of gene regulation.

Can be deployed quickly!

Can use multiple inputs or act as one of the multiple inputs.

Question 26

Ways sRNA works?

Answer 26

Inhibits or activate translation

Promote or prevent degradation

Promote post-translational processing

Alter the activity of other regulatory proteins

Question 27

How does sRNA activate translation?

Answer 27

Activate translation by binding to mRNA and changing a step loop conformation opening up an activator site

Question 28

How do sRNA promote processing?

Answer 28

Stabilize and in stable RNA long enough for RNAase III to come along and cleave it in the right spots

Question 29

An example of sRNAs altering regulatory proteins?

Answer 29

CsrB forms a stem-loop circle that binds regulatory protein CsrA (which has many jobs) so that it can’t activate or repress genes.

Question 30

In times of low iron what happens?

Answer 30

Fur a regulating proteinis released from two neighboring genes

1. Enterochelin (bind environmental iron)
2. sRNA which tags for degradation mRNA that codes for unnecessary high iron using proteins such as succinate dehydrogenase.

Question 31

Examples of first messengers?

Answer 31

Environmental signals- light, air, temperature, starvation, salinity

Question 32

Second messengers?

Answer 32

Ca2+, ppGpp, cAMP, c-di-GMP, NO, cGMP

Question 33

Effectors?

Answer 33

DNA, RNA, Protein complexes

Question 34

Outputs?

Answer 34

Motility, aggregation, PBS degradation, translation initiation

Question 35

ppGpp effects?

Answer 35

In stress or starvation, it acts on RNAP which in turn

1. Turns on stress genes/proteins and amino acid biosynthesis (many things) and
2. Turns off DNA replication, lipid synth, ribosomes elongation factors, and rRNA

all at once.

Question 36

c-di-GMP functions?

Answer 36

In post exponential growth/stationary phase

2 step process to make- DGC’s make it from GTP and PDE (phosphodiesterase) degrades to GMP

Turns on biofilm genes to make pili and turns off motility/flagella genes