L3 - Gene expression & introduction to Operons

Question 1

what is a genome?

Answer 1

The whole of the DNA in an organism, the set of genes AND non-coding sequences.

Question 2

what is an operon?

Answer 2

In bacteria, genes arranged into clusters of genes with related functions called operons. All genes in an OPERON are transcribed from one promoter to give a POLYCISTRONIC mRNA (polycistronic = ‘many genes’)

Question 3

what is a Shine-Dalgarno sequence?

Answer 3

In Bacteria ONLY, it is a ribosome binding site to which the 3’ end of rRNA binds to in mRNA near the start codon. This is to distinguish between AUG start codon starting the protein sequence and other methionine codons in the middle of protein sequence.

Question 4

how do eukaryotes differ from prokaryotes in relation to start codons and transcription?

Answer 4

In EUKARYOTES: genes transcribed individually, ribosome normally binds to beginning of mRNA and moves to FIRST AUG in mRNA
In PROKARYOTES: genes can be arranged into operons, Shine-Dalgarno sequences used to distinguish start codons

Question 5

what is a promoter in a gene?

Answer 5

A promoter provides a binding site for RNA polymerase and signals the start point and direction of transcription.
In SINGLE GENES = each gene has its own promoter
In OPERONS = one promoter per operon

Question 6

what are the two regions that identify most promoters in bacteria?

Answer 6

-35, -10 (numbering relative to position where transcription starts - which is normally +1)
these are CONSENSUS SEQUENCES: the bases shown are the most common but not invariable
(e.g. Shine-Dalgarno = consensus sequence)

Question 7

what is bacterial RNA polymerase’s structure and function?

Answer 7

The RNA polymerase core enzyme contains 5 polypeptides: 2 alpha copies, beta, beta’, w.
The core enzyme can synthesise RNA but DOES NOT recognise specific promoter sequences, Specific binding to promoters requires SIGMA FACTOR.
CORE ENZYME + SIGMA FACTOR = HOLOENZYME

Question 8

what is a HOLOENZYME?

Answer 8

core enzyme + sigma factor, meaning it can bind specifically to promoters, other enzymes cant without sigma factor.

Question 9

what is the process of bacterial transcription termination?

Answer 9

1. the end of mRNA folds into a STEM-LOOP STRUCTURE followed by a succession of uracils
2. this structure destabilises RNA-DNA interaction
3. RNA polymerase dissociates from DNA and transcription terminates

Question 10

when is a gene expressed?

Answer 10

A gene must be expressed at the correct level when its product is needed

Question 11

what specifies which genes are expressed in bacteria?

Answer 11

sequences within an operon specify when its genes are expressed.

Question 12

what is the Lac Operon function?

Answer 12

(an example of gene expression regulation in bacteria)
To allow E. coli to grow on lactose, a disaccharide formed from 1 glucose and 1 galactose. Galactose and glucose are ISOMERS - same formula C6H1206 but structures differ

Question 13

what are the two enzymes required by E. coli to metabolise lactose?

Answer 13

1. Lactose permease - transports lactose into the cell

2. B-galactosidase - hydrolyses lactose to glucose and galactose

Question 14

which 3 genes are transcribed together in the lac operon?

Answer 14

lacZ - codes for B-galactosidase
lacY - codes for lactose permease
lacA - codes for B-galactoside transacetylase

Question 15

which sugar does E.coli prefer to use?

Answer 15

prefers glucose, therefore the lac operon only transcribed when its gene products are needed, when glucose is absent.

Question 16

what would happen in scenario where lactose absent OR glucose is present?

Answer 16

lac operon would not be transcribed, fewer than 5 molecules of B-galactosidase per cell

Question 17

what would happen in scenario where lactose present and glucose absent?

Answer 17

lac operon would be transcribed, approx 5000 molecules of B-galactosidase per cell

Question 18

how are evidence of mechanisms expressed to explain how the cell recognises whether lactose is present or absent?

Answer 18

Majority of evidence comes from analysis of MUTANTS with altered gene expression. A simple way to detect mutants is to screen for B-galactosidase activity, on agar plates colour change from colourless to blue of artificial substrate X-gal can be seen. (colonies containing active B-galactosidase are blue)

Question 19

what regulates transcription in response to lactose?

Answer 19

a REGULATORY PROTEIN regulates transcription by one of 2 mechanisms:

1. regulatory molecule INCREASES transcription when lactose is PRESENT (activator)
2. reg. molecule DECREASES transcription when lactose is ABSENT (repressor)

Question 20

under what conditions would lac operon transcription be turned on? (provided glucose is absent)

Answer 20

1. in a normal cell
2. in a mutant where there is a non-functional ACTIVATOR protein.
3. in a mutant where there is a non-functional REPRESSOR protein

Question 21

how does the repressor repress?

Answer 21

the repressor binds to a specific DNA sequence in operon, named the OPERATOR.
there can be mutations in the operator that prevent the repressor from binding

Question 22

what causes the repressor to stop repressing?

Answer 22

there is a signal molecule that signals the presence of lactose, there are mutants where the signal molecule cannot bind to the repressor

Question 23

in a cell with a non-functional ACTIVATOR protein, when would lac operon be transcribed?

Answer 23

never - non inducible

Question 24

in a cell with a non-functional REPRESSOR protein when would the lac operon be transcribed?

Answer 24

all the time - constitutive