Microbiology Exam Ii (Slides 13-15) 326R

Front 1

What is an operon?

Back 1

a cluster of bacterial genes along with an adjacent promoter that controls the transcription of those genes.

Front 2

What is a operator?

Back 2

stretch of DNA, partially overlaps promoter and transcription start site

Front 3

What is the function of Beta galactosidase (LacZ)?

Back 3

cleave lactose into galactose and glucose, also catalyzes a side reaction of lactose to allolactose.

Front 4

What is the function of Lactose Permease (LacY) ?

Back 4

transports lactose across the cell membrane

Front 5

What is the function of Transacetylase (LacA) ?

Back 5

Function unknown

Front 6

What is the repressor in the Lac operon?

Back 6

The LacI is the repressor and this will stop transcription

Front 7

What can remove the lac I from the operator in the lac operon and why is this beneficial?

Back 7

The allalactose made by the LacZ can provide this function. This is beneficial because the lactose will go to form allactose, signaling there is still lactose and need to create other enzymes.

Front 8

In E. Coli what is prefered, Lactose or Glucose?

Back 8

Glucose

Front 9

Does the high levels of glucose increase or decrease the cAMP?

Back 9

Decrease

Front 10

What creates ATP to cAMP?

Back 10

Adenylate cyclase

Front 11

What does cAMP bind to in the lac operon structure?

Back 11

Binds to CAP

Front 12

What is the function of the CAP?

Back 12

Catabolite Activator Protein.The presence of CAP upstream of the promoter increases the affinity of RNA
polymerase to the Lac promoter – RNA transcription increased

Front 13

What happens when Lac operon is: Lactose High, Glucose Low?

Back 13

cAMP levels high, allowing promoter activation by CAP
Allolactose levels are high, lac repressor does not bind operator.
High levels of transcription

Front 14

What happens when Lac operon is: Lac low and Glu low

Back 14

allolactose levels low, lac repressor binds operator
cAMP levels high, CAP binds operator
Low levels of transcription

Front 15

What happens when Lac operon is: Glu High and Lac High

Back 15

Lac repressor will not bind operator at high levels since allolactose is present
cAMP levels will be low since glucose levels are high – promoter will not be activated by CAP
Low levels of transcription

Front 16

What happens when Lac operon is: Glu High and Lac low

Back 16

Lac repressor binds operator since allolactose levels are low.
cAMP levels are low, no promoter activation by CAP
Very little to no transcription.

Front 17

In the Trp operon, how many proteins are used to make ____ of enzymes to synthesis of Tryptophan?

Back 17

5;
3

Front 18

The Trp operon is halted under high or low levels of Trp?

Back 18

High Levels

Front 19

Lactose is an _____, tryptophan is a ______

Back 19

inducer;
repressor

Front 20

What are the five trp genes?

Back 20

trp E, trp D, trp C, trp B, and trp A

Front 21

What is the repressor for the trp operon?

Back 21

The trpR.
Trp will bind to trpR, change its conformation and repress the operon.

The binding of repressor to operator prevents transcription

Front 22

What are some genes that are advantageous in large concentrations of bacteria?

Back 22

Beta Lactamase and Luciferase and they will communicate via signialing molecules

Front 23

What produces the light in squids?

Back 23

Luciferase

Front 24

The Lux operon encodes several proteins such as:

Back 24

Luciferase, which produces light, and all the enzymes necessary for making VAI

Front 25

When there is a high density of VAI what happens?

Back 25

VAI will bind Lux protein, changing its conformation such that VAI will now bind at the LuxO site. Binding of Lux to LuxO increases recruitment of RNA polymerase to the promoter. This will increase the production of luciferase and VAI synthesis genes.

Front 26

What happens at low populations of VAI?

Back 26

Low population density:
VAI diffuses out of cell,
Cytoplasmic VAI levels remain low

Front 27

In the attenuator sequence for the Trp operon, what RNA regions are complementary?

Back 27

Regions
21,23
32,34

Front 28

Of the sequences in the attenuator, which causes rho independent termination and which doesn't (forms a premptor)?

Back 28

Preemptor-23
Rho independent terminator-34

Front 29

Describe how the TRANSLATION is regulated via the attenuator in trp?

Back 29

Well the Ribosome will bind to the Shine Dalgarno RBS and let translation begin.

-Usually it should be noted that the leader peptide usually has no known function and is degraded quickly

Translation will occur but if there is a low tRNA charged with trp, then the 1 attenuator can't bind with 2 and the 2-3 premptor sequence is made.

If there is charged tRNA then the Ribosome will make it to the 1 and the 1-2 will bind and then the 3-4 Rho independent sequence will form.

Front 30

What is more effective direct repression of trp or indirect?

Back 30

Direct(trp +trpR repressor binds to trp operator)

Front 31

How many trp sequences are in region 1 of the attenuator sequence?

Back 31

The leader peptide sequence
contains two consecutive trp codons within region one.

Front 32

polycistronic mRNA with multiple independent translation units (ORFs),
each with its own ______-______ ___ , start and stop codons.

Back 32

Shine-Delgarno RBS

Front 33

What is unique for the regulation of beta lactamase?

Back 33

The Rho-independent terminator is formed if there is low levels of ribosomes and not formed if there is high levels of Ribosome

Front 34

Describe the HutP in inhibiting or allowing termination?

Back 34

If there are low levels of Histidine then the HutP is not able to bind to RNA and the stem loop will be formed in RNA and allow termination.

If there are high levels of Histidine this will allow 6 Histidines to bind to HutP and Hut P will then bind to RNA. This binding will be in proximity to the stem loop and not allow termination to occur.
-Then the Histidine utilizing enzymes will be created

Front 35

How many histidine enzymes did we discuss?

Back 35

Five enzymes for Histidine breakdown and utilization

Front 36

Is this a DNA or RNA?
Is often synthesized, recycled and degraded.

Back 36

RNA

Front 37

Pick the wrong description concerning RNAse A:
It is a ubiquitous enzyme
It is not stable
Is double strand specific

Back 37

It is highly stable
It is single strand specific but will degrade ds RNA given time

Front 38

What is the underlying reason why RNA is more susceptible to attack from the RNAse in contrast to DNA?

Back 38

2’ OH group on ribose can attack nearby Phosphate this is the mechanism underlying base hydrolysis and some RNAses.

Front 39

Explain the base hydrolysis of RNA?

Back 39

The 2'OH will attack a nearby active Phosphorus. This happens because the Phosphorus is electro-positive and attracts the negatively
charged oxygen

This will then form a 2’ 3’ cyclic phosphate.

Front 40

What are the structural RNAs that are not translated?

Back 40

snRNA (small nuclear RNA)
-Splicing factors in eukaryotes
tRNA
rRNA

Front 41

What are the regulatory RNA's?

Back 41

siRNA
-antiviral defense mechanism


RNAi
-Long (50bp to a few kb) ds RNA, causes degradation of complementary mRNA
-Causes sequence-specific hydrolysis of target RNA

microRNA-Eukaryotes
-Repress translation

small RNA's-Prokaryotes
-ssRNA in bacteria, 20-300 nt long
-Mediation by Hfq
-Regulation may be positive or negative
-May work by binding directly to target mRNA or by binding a regulatory protein
-DsrA, CrsA, RhyB

Front 42

Describe small RNA's?
Euk or Prok, function, action

Back 42

The small RNA's attack Prokaryotes and act by regulating translation of RNA, (either positive or negative) and acts by base pairing with mRNA.

Front 43

What is the function of Hfq?

Back 43

This is a RNA chaperone, proteins that assist the non-covalent folding or unfolding and the assembly or disassembly of other macromolecular structures, it Binds and stabilizes (extends half-life) small RNAs, including DsrA

Front 44

DsrA is an 85nt ____

Back 44

sRNA

Front 45

When Hfq binds to DsrA what happens?

Back 45

This protects the DsrA from RNAse but still allowing DsrA to base-pair with its target RNAs

Front 46

What does DsrA function as?

Back 46

DsrA may upregulate or downregulate expression of genes, by increasing or decreasing the rate of translation.

Front 47

What genes does DsrA regulate, by increasing or decreasing the rate of translation?

Back 47

sigmaS, iron scavenging genes, virulence factors and biofilm forming genes

Front 48

In the absence of Hfq what can one expect to the function of DsrA?

Back 48

The effectiveness of DsrA regulation is reduced

Front 49

Describe how DsrA-Hfq complex decreases and increases translation?

Back 49

Increase translation:
mRNA forms a hairpin at the 5'end and this will decrease translation.
The DsrA-Hfq complex will bind.
DsrA base-pairs to one stem of hairpin, causing RBS to be single
stranded, allowing binding of ribosome and increasing translation

Decrease:
The mRNA is working fine and then all of a sudden the DsrA-Hfq complex base pairs just past or near start codon and inhibits movement of ribosome.
Translation is stopped. Expression of protein dramatically reduced

Front 50

What does CrsA do to mRNA?

Back 50

CrsA will bind the 5’ end of some (not all) mRNAs,
covering up the RBS and inhibiting translation.

CsrA is a sequence–specific RNA binding protein

Front 51

What does CrsB do to mRNA?

Back 51

CsrB is an sRNA.
CsrB has multiple binding sites for CsrA.
It competes with mRNAs for CsrA binding. Binding CsrB RNA
to CsrA RNA allows ribosome assembly and initiation of translation.

The relative ratio of CsrB, CsrA and the target mRNA
determines if the mRNA is translated or not:
High levels of mRNA and CsrB favor translation,
higher levels of CsrA inhibit translation.

Front 52

How can iron be detected within the bacteria?

Back 52

Iron will bind to Fur

Front 53

Describe what happens when Fur (transcription factor) binds to iron?

Back 53

The iron will bind to Fur and then Fur will then bind to RyhB,a 90nt sRNA.
RyhB will then form a complex with Hfq

The RyhB RNA /Hfq complex will then activate RNAse E.

-This RNase activity is specific for mRNAs which encode iron-using genes (including Krebs cycle and ETC genes).

Front 54

RyhB is typically expressed at _____ levels during the stationary phase and at ______ levels during the log phase

Back 54

High
Low

Front 55

When Fur is bound to iron, it ______ transcription of RyhB. RyhB is a 90nt sRNA

Back 55

represses

Front 56

What complex, other than DsrA, forms a complex with Hfq?

Back 56

RyhB

Front 57

What system is used to help misfolded proteins?

Back 57

GroES/GroEL COMPLEX

Front 58

What are the mammalian equivalents to the GroES/EL?

Back 58

HSP 60 and HSP 10.

Front 59

Which is bigger GroES or EL?

Back 59

GroEL is a protein of about 60 kilodaltons

GroES is a protein of about 10 kilodaltons (small).

Front 60

Describe how GroES/EL attempt to fix misfolded proteins?

Back 60

The misfolded protein will have a large hydrophobic portion showing and will bind to the interior of the GroEL, GroEL forms a barrel shaped structure composed of two rings of seven polypeptides.

The binding of the misfolded protein ONLY occurs after ATP has bound to GroEL and changed its structure.

GroES will then form a cap over the GroEL

ATP bound to the GroEL and this will attempt to fix the misfolding in the protein.

GroES is then released along with the protein.
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