kevin (L1-4) Flashcards
Prokaryotic molecular Biology Transcription and the control of gene expression
constitutive expression
genes that are always expressed
general transcriptional mechanisms
these are the mechanisms used for the control of gene expression. there are 2:
INDUCTION - the switching on of genes when they are required
REPRESSION - the switching off of genes when they are not required
operon
many genes being transcribed together from a single promoter
An operon is regulated like it was a single gene
Have a linked function and produce a polycistronic mRNA (where more than one gene is expressed in the same mRNA strand)
repressors
Repressors are regulatory proteins which prevent transcription when bound to the DNA
(Regulatory proteins are often converted between inactive and active states by binding of small molecules (effectors like inducers and co-repressors))
activators
Activators (apoinducers) are regulatory proteins which activate transcription when bound to the DNA
(Regulatory proteins are often converted between inactive and active states by binding of small molecules (effectors like inducers and co-repressors))
regulons
Genes associated with a particular physiological function may not be in just one operon
These operons may be controlled by a single regulatory protein – together they are called a REGULON
The phosphate or PHO regulon consists of >80 genes in many operons, all are controlled by one regulatory protein
global control / global regulation system
often an organism needs to regulate many genes associates with different metabolic functions in response to a single environmental factor.
Control systems that operate on such a wide basis are known as GLOBAL CONTROL or GLOBAL REGULATION SYSTEMS
diauxic growth
If both glucose and an alternative carbon source is available the glucose is used first - Diauxic growth (when 2 carbon sources are being utilised in succession not at the same time)
catabolite repression
Glucose represses the synthesis of enzymes that metabolise less preferred carbon sources
lac phase in an e coli’s diauxic growth
When the cells run out of glucose there is a very rapid induction of the enzymes of lactose metabolism. We enter the lac phase, and the e coli switches its genes and proteins it’s producing to utilise the second carbon source, lactose
lac Z
gene that encodes β-galactosidase, which cleaves β-galactosides into monosaccharides
lac Y
gene that encodes β-galactoside permease, a cytoplasmic membrane protein which transports β-galactosides into the cell
laz A
gene that encodes β-galactoside transacetylase, which is thought to detoxify toxic β-galactosides by acetylation
promoter
lacP, where RNA polymerase bind
operator
lacO, where the lac repressor binds
cap site
where the cAMP acceptor protein (CAP; also called the catabolite repression protein, CRP) binds
adenyl cyclase
ATP is converted to cAMP by the enzyme adenyl cyclase
When glucose is present adenyl cyclase is inactive
Once glucose is used up adenyl cyclase becomes active and makes cAMP
Insertion vectors
Non-essential DNA has already been removed at a specific restriction site and DNA can be cloned and inserted into this site
Replacement vectors
Non-essential ‘stuffer’ DNA is replaced with new non-coding DNA to be cloned
fosmids
fosmids are large plasmids based on the F plasmid with cos-sites, lacZ and a T7 promoter added. (they can be used for us to fit in a lot of dna).
The cos-sites allow the vector to be packaged into λ phage particles for transfection into E. coli
Up to 40kbp (+8kbp of vector) can be packaged into each phage particle
plasmid/phage cloning mix
Once a fosmid is transfected into an e coli cell, they are maintained in the cell as a plasmid.
This is a mix between plasmid and phage cloning.
fosmids and cosmids
If you remove everything but its essential parts, you can create a plasmid vector (fosmid or cosmid) that allows you to produce bigger fragments of dna. These are engineered molecules (originally come from an F plasmid) that were engineered to have cos sites (at the end of a lambda phage, which allow it, when the dna is put into an e coli cell, to circulise itself).
So the 2 cos sites bind to each other to produce a very large 50kbp plasmid.
ti plasmid
The Ti plasmid is found in the plant pathogen Agrobacterium tumefaciens and causes tumours (crown galls) on plants
Only a specific portion of the Ti plasmid is transferred into the plant cell and is then integrated into the plant genome to damage those cells
BACs
BACTERIAL ARTIFICIAL CHROMOSOMES (BACs) – can carry up to 300 kbp of cloned DNA
Some vectors that can take very large fragments have been dubbed “artificial chromosomes”
(Based on the F plasmid they often carry selectable markers and their screenable marker is the lacZ gene)
