Regulation and Signaling

Question 1

What are the two main categories of protein regulation?

Answer 1

-Protein activity: Activate or inactivate a protein that’s already made
-protein amount: control the synthesis or degradation of a protein

Question 2

Examples of protein Activity regulation

Answer 2

-allosteric inhibition
-covalent modification
(phosphorylation/methylation, cleavage)

Question 3

Examples of protein amount regulation

Answer 3

-transcriptional regulation and attenuation (lac and trp operons)
-mRNA degradation
-repression of translation
-proteolysis
-any step along gene expression

Question 4

What is a Kinase?

Answer 4

an enzyme that transfers a phosphate to an acceptor molecule

Question 5

What is a phospatase?

Answer 5

An enzyme that removes phosphates

Question 6

An example of
transcriptional
repression,
a type of regulation
of protein amounts

Answer 6

-The lac operon
-encodes enzymes for lactose utilization (LacZ, Y, A)
- amounts of these enzymes is regulated because lactose is not a common nutrient for E.coli

Question 7

Methods of regulating protein amounts

Answer 7

-every step from the gene to the functional protein could be regulated
- the type of regulation involved varies from gene to gene
-often multiple types of regulations are involved

Question 8

What is attenuation?

Answer 8

-Premature termination of transcription
-attenuation regulates other amino acid biosynthesis operons in addition to the trp operon

Question 9

What do sRNAs do?

Answer 9

Inhibition of translation
-have a hair loop structure

Question 10

What happens when trp is scarce?

Answer 10

-There is no attenuation
- The ribosome stalls but doesn’t stop bc Trp is scarce
-anti terminator on tRNA forms
-RNA polymerase will continue transcribing
-trpE-A is expressed

Question 11

What happens when trp is abundant?

Answer 11

-attenuation occurs
-tRNA charged with trp are abundant so ribosome doesn’t stall
-terminator forms
- RNA polymerase stops transcribing
-trpE-A is not expressed

Question 12

What are the two important properties of the transcript of trpL?

Answer 12

1. can form different stem loop structures; terminator or anti terminator
2. has 2 adjacent tryptophan codons (UGG-UGG)

Question 13

Where is the trpL located?

Answer 13

Upstream of trpE-A in genome

Question 14

When does attenuation occur?

Answer 14

occurs when excess tryptophan is present

Question 15

What is a regulon?

Answer 15

a set of operons controlled by a common regulator

Question 16

What is a modulon?

Answer 16

-A set of regulons controlled by a common regulator such as (CAP-cAMP for glucose mediated catabolite repression)

Question 17

Globular Regulation

Answer 17

-catabolite repression
- a common regulator controls transcription of many different catabolic
enzymes, so bacteria selectively use their preferred energy source first

Question 18

CAP-cAMP

Answer 18

-binding of CAP-cAMP to regulatory region helps RNAP to bind, activating
transcription of catabolic enzymes (CAP = catabolite activator protein)
-high cAMP levels needed for CAP-cAMP binding
-enhances transcription of operons already induced, e.g. lac
operon by lactose
-CAP binding is sensitive to
cellular cAMP levels
-low glucose levels result in high
cAMP levels
-basis for “glucose effect” in many
bacteria = repression of
catabolism of other carbon
sources when glucose is present

Question 19

Signaling

Answer 19

Bacteria sense and respond to their environment

Question 20

Stress

Answer 20

=change
- to ability to cope with stress is critical to a microbes survival

Question 21

Two component Regulatory Systems

Answer 21

–highly conserved and
ubiquitous in bacteria
(also in fungi, plants)
-sensor kinases and
response regulators have
conserved amino acid
sequence motifs – easy
to annotate in the
genome
-absent from animals and
humans and important
for bacterial survival;
thus a great potential
drug target

Question 22

Two-component regulatory systems - examples from rhizobia

Answer 22

Rhizobia are root-associated bacteria that “fix” nitrogen in a symbiosis
with plants. Rhizobia convert dinitrogen gas into a form usable by plants.
Fixed nitrogen is critical for agricultural productivity.

Question 23

Nitrogen-fixing bacteria and legumes

Answer 23

• establishment of the symbiosis involves infection of plant cells in the root
  by the bacteria (which act as endosymbionts in the nodule).
  *the plant receives fixed nitrogen from the bacteria while the bacteria receive
  carbon sources from the plant

Question 24

Two-component regulatory systems - examples from rhizobia
low [O2 ]
FixL
FixJ
Genes encoding enzymes for nitrogen fixation

Answer 24

FixL/J in Sinorhizobium
meliloti is a two-
component system that
controls transcription in
response to O 2
concentration.
*FixL/J activates
transcription of the gene
for nitrogenase, which
catalyzes nitrogen fixation
when [O 2 ] is low.
*Nitrogenase enzyme is
inactive when [O 2 ] is high,
so it would be a waste to
make it then.

Question 25

Two-component regulatory systems - examples from rhizobia
?
ExoS
ChvI
Genes encoding enzymes for polysaccharide production

Answer 25

*ExoS/ChvI is a two-
component system in
Sinorhizobium meliloti
that controls transcription
in response to an
unknown signal.
*ExoS/ChvI activates
transcription of genes for
polysaccharide
production.
*The right amount of
polysaccharide is needed
to form the nitrogen-fixing
symbiosis with plants.

Question 26

Main points of lac operon

Answer 26

⇰ Glucose is present, Lactose is absent:
No transcription of the lac operon occurs. that’s b/c the lac repressor remains bound to the operator and prevents transcription by RNA pol. Also, cAMP levels are low b/c glucose levels are high, so CAP is inactive and cannot bind DNA.

⇰ Glucose is present, Lactose is present:
Low-level transcription of the lac operon occurs. the lac repressor is released from the operator b/c the inducer (allolactose) is present. cAMP levels, however, are low b/c glucose is present. Thus, CAP remains inactive and cannot bind to DNA, so transcription only occurs at a low, entry level.

⇰ Glucose is absent, Lactose is absent:
No transcription of the lac operon occurs. cAMP levels are high b/c glucose levels are low, so CAP is active and will bind to the DNA. However, the lac repressor will also be bound to the operator due to the absence of allolactose. This acts as a roadblock to RNA pol, preventing transcription.

⇰ Glucose is absent, Lactose is present:
Strong transcription of the lac operon occurs. the lac repressor is released from the operator b/c the inducer, allolactose, is present. cAMP levels are high b/c glucose is absent, so CAP is active and bound to the DNA. CAP helps RNA pol bind to the promoter, permitting high levels of transcription.

Takeaway:
The lac operon will be expressed at high levels if 2 conditions are met:

⇰ Glucose must be unavailable: when glucose is absent, cAMP binds to CAP, making CAP able to bind to DNA. Bound CAP helps RNA Pol attach to the lac operon promoter.

⇰Lactose must be available: if lactose is available, the lac repressor will released from the operator via binding of allolactose. this allows RNA pol to move forward on the DNA and transcribe the operon.