Lecture 8 - Bacterial Transcription & The Lac Operon

Question 1

what is sigma factor?

Answer 1

sigma factor is a polymerase subunit that recognises and binds to the promoter sequences on the DNA

Question 2

what happens when the sigma factor and the RNA polymerase combine?

Answer 2

they become the holoenzyme

Question 3

what does the binding of the sigma factor to the DNA then allow?

Answer 3

allows the RNA polymerase to recognise the transcription start site and bind strongly to the DNA

Question 4

what do the different types of sigma factors allow for?

Answer 4

they different types allow for the different groups of genes to be regulated

Question 5

what is the most common sigma factor and what is it responsible for?

Answer 5

sigma 70 is the most abundant sigma factor and its expressed all the time in being responsible for the transcription of housekeeping genes

Question 6

housekeeping genes:

Answer 6

housekeeping genes are genes that are critical for cell survival and therefore covers the majority of genes

Question 7

what can be found around the promoter and what do they do?

Answer 7

they are regulator sites around the promoter that can inhibit or enhance transcription

Question 8

what is the operator site?

Answer 8

sequence recognised by repressor genes and upon binding physically blocks RNA polymerase from binding

Question 9

activator sequences:

Answer 9

activator sequences bind to proteins that can help to increase the efficiency of the RNA polymerase to bond to the DNA

Question 10

sigma factor with the holoenzyme initiates:

Answer 10

sigma factor with the holoenzyme initiates the “melting” of the DNA helix, separating the strands

Question 11

the specific promoter sequence that the sigma factor has bound to is only on one strand so…

Answer 11

… this is what determines whether the top or bottom strands will be transcribed

Question 12

in bacterial transcription nucleotides replicate 5’-3’ just like in DNA replication, but with ribonuleotides being added instead:

Answer 12

thymine is replaced with uracil

Question 13

what happens as the RNA transcript is being created?

Answer 13

the sigma factor dissociates from the RNA polymerase as it is no longer needed - sigma factor now free to bind to another RNA polymerase elsewhere

Question 14

RNA polymerase transcribes through to the terminator sequences which creates…

Answer 14

… a hairpin structure which causes the RNA polymerase to stall and combined with the stretch of uracils on the end of the transcript, there is reduced interaction between the DNA, RNA, and the RNA polymerase - this causing the termination of transcription and release of RNA transcript

Question 15

where is DNA in bacteria?

Answer 15

cytoplasm

Question 16

how can transcription and translation take place in bacteria?

Answer 16

transcription and translation can occur simultaneously and means that the RNA transcript is generally not modified

Question 17

The binding of sigma factor to the RNA polymerase for it to recognise the promoter sequence is critical for…

Answer 17

…bacterial transcription to occur, which can be affected by DNA-binding proteins binding to activators or repressors on the DNA

Question 18

once the RNA polymerase has melted the DNA strands the…

Answer 18

… polymerase can begin to transcribe the DNA into RNA, at which point the sigma factor disassociates

Question 19

transcription continues until it reaches the termination sequence where the combination of the…

Answer 19

… RNA hairpin and the AT-rich region results in transcription ending

Question 20

how do operons allow efficient response to environmental conditions in bacteria?

Answer 20

because they’re single celled organisms they need tight transcription control to ensure efficient use of energy and resources - operons allow for a coordinated response to achieve this

Question 21

what are operons?

Answer 21

operons are polycistronic genes allowing for coordinated expression of multiple genes at the same time, they are controlled by the same promoter

Question 22

what is the Lac Operon?

Answer 22

a well defined model of regulating gene expression, by looking at it we can get a full appreciation of how gene expression is regulated in bacteria at the level of transcription

Question 23

what genes control lactose metabolism?

Answer 23

multiple genes from the Lac Operon are involved in the metabolism of lactose intolerance glucose and galactose

Question 24

baseline state of the Lac Operon:

Answer 24

by default the Lac Operon is “turned off” through the Lac repressor coded lac-I gene, this occurs when the cell has all the glucose it needs

Question 25

what is the lac-I gene considered to be?

Answer 25

the lacI gene is considered a trans-regulator of the Lac Operon, it has its own promoter and terminator sequence

Question 26

when is the lac repressor transcribed and expressed?

Answer 26

always

Question 27

allostery:

Answer 27

additional molecules bind to the DNA-binding proteins and causes a conformational change that alters how it binds to the DNA, this allows for activation or inhibition of gene expression in response to stimuli

Question 28

how does the repressor keep Lac Operon “turned off”?

Answer 28

the repressor binds to the operator in the Lac Operon preventing RNA polymerase from binding

Question 29

effects of allosteric regulation:

Answer 29

molecules can induce different effects dependent on the DNA binding proteins:

–Inducible repressors
•Do not inhibit transcription when bound to effector molecule

–Repressible repressors
•Inhibit transcription when bound to effector molecule

–Repressible activators
•Do not activate transcription when bound to effector molecule

–Inducible activators
•Cause transcription when

Question 30

what happens when the cell runs out of glucose?

Answer 30

when the cell runs out of glucose, then the cell produces cyclic AMP from breaking down ATP

Question 31

what happens once cyclic-AMP has been produced?

Answer 31

This cyclic AMP can then bind to the protein cAMP-bound catabolite activator protein (CAP)

Question 32

as cyclic AMP acts as an inducible activator of CAP, this can then…

Answer 32

bind to the CAP binding site upstream of the Lac Operon promoter

Question 33

if there is no lactose for the cell to convert into glucose then…

Answer 33

… the repressor remains bound to the DNA to prevent CAP from helping to bind RNA polymerase

this is because there is no point wasting further energy making enzymes when there is no lactose to break down

Question 34

if lactose is present in the cell:

Answer 34

this will be metabolised into allolactose by β-galactosidase

there is always a small amount of β-galactosidase present in the cell

Question 35

action of β-galactosidase:

Answer 35

acts as a detector system for the presence of lactose that the cell can use

Question 36

what does allolactose act as in bacteria?

Answer 36

allolactose will act as an inducible repressor and bind to the Lac repressor protein, causing it to undergo a conformation change and not be able to bind to the operator site

Question 37

what will the RNA polymerase with the sigma factor be able to do once the lac repressor protein has been removed off of the operator?

Answer 37

RNA polymerase with the sigma factor will then be able to bind to the DNA with the assistance of CAP, transcription will then occur, creating an RNA which which will be turned into protein - allowing lactose to be transported into the cell to be broken down into glucose

Question 38

returning to baseline expression:

Answer 38

as cyclic-AMP generation reduces since glucose becomes the main source of carbon and energy in the cell, this means there will be less to bind to CAP, meaning less CAP will be able to bind to DNA - ensuring the cell is not continually making more enzyme when there’s already lactose & glucose in the cell which is already being converted as needed

Question 39

Lac Operon Restarting:

Answer 39

eventually the levels of lactose and allolactose will drop, and the cycle will restart with the binding of the Lac repressor back on to the operator

Question 40

the Lac Operon has an elegant way to regulate the expression of the genes involved in metabolising lactose to glucose:

Answer 40

using both a repressor and activator, this ensures that this is down in an efficient manner when required without wasting energy making gene products that are not going to be used

Question 41

Tryptophan Operon:

Answer 41

• tryptophan also has an operon where there are five genes within it related to tryptophan biosynthesis

• the levels of tryptophan regulate the binding of the active repressor to the operator site within the promoter region

• when there are high levels, it acts as a repressible repressor and turns off the operon

Question 42

what is maltose an example of?

Answer 42

maltose is an example of the molecule regulating its own metabolism with the maltose operon

Question 43

operons allow for efficient:

Answer 43

regulation of genes related on the same pathway

Question 44

gene expression from the operons can be modulated by signals from the pathways they are involved in:

Answer 44

allosteric regulation of the regulatory proteins involved in enhancing or inhibiting transcription

Question 45

what happens when there are high levels of maltose?

Answer 45

when there are high levels of maltose, this binds to the maltose activator protein itself, which allows for the expression of the genes for maltose metabolism

when this reduces, there is less and less to bind to the activator protein, and transcription and gene expression of the operon is reduced