H6
specialized expression vectors have been created that provide genetic elements for controlling
transcription, translation, protein stability, and secretion of the product of the cloned gene from the
host cell. They manipulated to modulate gene expression include the promoter and transcription
terminator sequences, the strength of the ribosome-binding site, the number of copies of the cloned
gene and whether the gene is plasmid borne or integrated into the genome of the host cell, the final
cellular location of the synthesized foreign protein, the efficiency of translation in the host organism,
and the intrinsic stability within the host cell of the protein encoded by the cloned gene.
Gene Expression from Strong and Regulatable Promoters
The minimal requirement for an effective gene expression system is the presence of a strong and
regulatable promoter sequence upstream from a cloned gene. A strong promoter is one that has high
affinity for RNA polymerase. The ability to regulate a promoter enables the cell (and the researcher)
to control the extent of transcription in a precise manner. The promoter lac (lactose) operon of E. coli
has been used extensively for expressing cloned genes.
Plasmid instability is a major problem that may prevent the efficient production of a plasmid-borne
gene product on a large scale. To overcome this, it is needed to control transcription in such a way
that a cloned gene is expressed only at a specific stage in the host cell growth cycle. This can be
achieved by using a strong regulatable promoter, by using expression vectors.
Regulatable Promoters
The most used strong regulatable promoters are lac and trp (tryptophan) operons; the tac promoter.
Each of these promoters interacts with regulatory proteins (repressors), which provide a controllable
switch for either turning on or turning off specific transcription of adjacent cloned genes. Each of
these promoters is recognized by the major form of the E. coli RNA polymerase holoenzyme. This
holoenzyme is formed when a protein, called sigma factor, combines with the core proteins of RNA
polymerase. Sigma factor directs the binding of the holoenzyme to promoter regions on the DNA.
In the absence of lactose in the growth medium, the E. coli lac promoter is repressed (turned off) by
the lac repressor protein, which prevents the lac operon from being transcribed. Induction (turning
on) of the lac promoter is achieved by the addition of either lactose or IPTG to the medium(IPTG
heeft namelijk dezelfde structuur als lactose). prevents the lac repressor from binding to the lac
operator. The enzyme β-galactosidase is encoded by the lacZ gene of the lac operon, and it is
involved in the cleavage of lactose into glucose and galactose.
Transcription from the lac promoter is also regulated by binding of the catabolite activator protein
(CAP) to a region of the DNA (the CAP box) upstream of the promoter region. When CAP binds to the
CAP box, it increases the affinity of the promoter for RNA polymerase so increasing transcription.
When inducer (lactose or IPTG) is present and there is no repressor bound to the operator, a high
intracellular concentration of cAMP can lead to a high level of transcription of the genes downstream
of the lac promoter.
The trp promoter regulates transcription of for tryptophan. This strong promoter is negatively
regulated (turned off) by the trp repressor protein complexed with tryptophan, which binds to the
trp operator and prevents transcription of the trp operon. Derepression (turning on) of the trp
promoter is achieved by removing tryptophan or by adding 3-indoleacrylic acid to the growth
medium.
specialized expression vectors have been created that provide genetic elements for controlling
transcription, translation, protein stability, and secretion of the product of the cloned gene from the
host cell. They manipulated to modulate gene expression include the promoter and transcription
terminator sequences, the strength of the ribosome-binding site, the number of copies of the cloned
gene and whether the gene is plasmid borne or integrated into the genome of the host cell, the final
cellular location of the synthesized foreign protein, the efficiency of translation in the host organism,
and the intrinsic stability within the host cell of the protein encoded by the cloned gene.
Gene Expression from Strong and Regulatable Promoters
The minimal requirement for an effective gene expression system is the presence of a strong and
regulatable promoter sequence upstream from a cloned gene. A strong promoter is one that has high
affinity for RNA polymerase. The ability to regulate a promoter enables the cell (and the researcher)
to control the extent of transcription in a precise manner. The promoter lac (lactose) operon of E. coli
has been used extensively for expressing cloned genes.
Plasmid instability is a major problem that may prevent the efficient production of a plasmid-borne
gene product on a large scale. To overcome this, it is needed to control transcription in such a way
that a cloned gene is expressed only at a specific stage in the host cell growth cycle. This can be
achieved by using a strong regulatable promoter, by using expression vectors.
Regulatable Promoters
The most used strong regulatable promoters are lac and trp (tryptophan) operons; the tac promoter.
Each of these promoters interacts with regulatory proteins (repressors), which provide a controllable
switch for either turning on or turning off specific transcription of adjacent cloned genes. Each of
these promoters is recognized by the major form of the E. coli RNA polymerase holoenzyme. This
holoenzyme is formed when a protein, called sigma factor, combines with the core proteins of RNA
polymerase. Sigma factor directs the binding of the holoenzyme to promoter regions on the DNA.
In the absence of lactose in the growth medium, the E. coli lac promoter is repressed (turned off) by
the lac repressor protein, which prevents the lac operon from being transcribed. Induction (turning
on) of the lac promoter is achieved by the addition of either lactose or IPTG to the medium(IPTG
heeft namelijk dezelfde structuur als lactose). prevents the lac repressor from binding to the lac
operator. The enzyme β-galactosidase is encoded by the lacZ gene of the lac operon, and it is
involved in the cleavage of lactose into glucose and galactose.
Transcription from the lac promoter is also regulated by binding of the catabolite activator protein
(CAP) to a region of the DNA (the CAP box) upstream of the promoter region. When CAP binds to the
CAP box, it increases the affinity of the promoter for RNA polymerase so increasing transcription.
When inducer (lactose or IPTG) is present and there is no repressor bound to the operator, a high
intracellular concentration of cAMP can lead to a high level of transcription of the genes downstream
of the lac promoter.
The trp promoter regulates transcription of for tryptophan. This strong promoter is negatively
regulated (turned off) by the trp repressor protein complexed with tryptophan, which binds to the
trp operator and prevents transcription of the trp operon. Derepression (turning on) of the trp
promoter is achieved by removing tryptophan or by adding 3-indoleacrylic acid to the growth
medium.
