Gene Regulation

in Prokaryotes
The operon in prokaryotes
 Operon is a linear arrangement of genes whose
activities are coordinately regulated
 In an operon, there are many genes which are
transcribed into one long mRNA from which each of
the proteins are translated separately. Whole operon
is regulated by a single promoter. Thus all the genes
in an operon are coordinately regulated. But in
eukaryotes, each mRNA gives rise to a single
polypeptide or a protein molecule.
 Operon in Escherichia coli (lac-operon) has half life 3
min.
 The classical study of Jacob and Monod in 1961 on
the lactose (lac) operon in E. coli is very important in
understanding the regulation of genes in prokaryotes.
Genes of lac Operon

 The lac operon consists of three structural

genes for three proteins:
 1) β-galactosidase: which hydrolyzes lactose into
glucose and galactose.
 2) Lactose permease: it is a transporter for the
uptake of lactose into the cell
 3 Thiogalactosidase transacetylase is involved in
the detoxification of non-metabolizable b
galactosides which can be secreted from the cell.
Component of the lac operon
 cAMP = Cyclic AMP, signal molecule formed from ATP by the
action of adenylate cyclase.
 CAP /CRP = Catabolite activator protein / Catabolite receptor
protein. This protein is regulated by cAMP. When glucose is
absent cAMP conc. increases and it binds to CRP. This cAMP –
CRP complex binds to promoters which activate operons.
 Inducer = Lactose
 Repressor = A protein encoded by the lacI gene.
 DNA directed RNA polymerase - means for transcription
 Regulatory sequences = promoter and operator where RNA
polymerase and repressor bind
 the genes
 lac I = Repressor

 lac Z = β-galactosidase - lactose  glucose + galactose

 lac Y = Permease – Transport of lactose

 Lac A = Transacetylase – detoxification of non-metabolized β-
galactosidase from the cell
Function of lac operon
 Under normal conditions, E. coli utilizes glucose, a
readily available carbon source. Lactose is an inferior
carbon source and is not usually available to the
bacterium. Hence in the absence of lactose or presence
of glucose, the lac operon is dormant or is in a repressed
state. A specific regulatory gene (lac I) is involved in this
repression which produces a repressor protein. There
are two domains on this repressor domain. One has an
affinity for the lac operator which is the DNA sequence of
the operon and the other domain binds an inducer
molecule, in this case lactose. Lactose - free repressor
binds to the operator and prevents transcription by RNA
by RNA polymerase. However, in the presence of
lactose when lactose forms a complex with the repressor
it can no longer bind to the operator and the lac operon
mRNA is transcribed.
The Promoter
 It is the site where RNA polymerase physically binds
to genes. In the lac operon, the promoter is slightly
upstream of the operator site. Binding of the repressor
interferes with RNA polymerase binding, thereby
preventing transcription. The DNA sequence of a
promoter determines the maximum possible rate of
gene transcription. In E. coli promoters there are two
separate sets of nucleotide sequences which are
highly conserved and therefore essential for
transcription. If the transcriptional start site is
designated +1 base position, at -35 there is the RNA
polymerase recognition and binding site. At -10 the
double – stranded DNA is thought to be open for
transcription of the mRNA using the DNA
complementary minus strand as the template.
Transcription is in the 5 to 3 prime direction.
Consensus sequences of promoter

 These are RNA polymerase bindings sites

 The consensus sequence of a particular functional component of a
gene is a DNA sequence that appears to be conserved between
different genes with only minor changes. Consensus sequence tends to
occur in a group of closely related genes. Hundreds of promoter regions
have been sequence in E. coli and only the -35 and -10 regions exhibit
consensus sequences. These RNA polymerase binding sites have the
consensus sequences TTGACA at -35 and TATAAT at the -10 box
(Pribnow box). The distance b/w these two consensus sequences is 16-
18 bp with some exceptions as low as 15 or as high as 20 bases. This
distance is critical because of the interaction b/w the DNA strand and
RNA polymerase.
 In few bacterial promoters, one of the consensus sequences is missing
and supporting proteins are required to initiate transcription. The site for
initiation of RNA polymerase activity i.e. the start base for transcription
is usually a purine (A or G). Commonly it is the centre of the sequence
CAT but it is not essential in all cases.
 (negative positive regulation of (AMP)
Regulation of activity
 The turning off of the lac operon caused by repressor binding to the
operator is a prime example of negative genetic control. Bacteria also
require +ve regulation of gene transcription for which a transcriptional
activator is required. The best example of +ve regulation is by cyclic
AMP (cAMP). In the absence of glucose the concentration of cAMP
becomes high and it binds to CRP (cAMP receptor protein) a DNA
binding protein. This complex in turn binds to promoters which activate
operons.
 cAMP is universally distributed in all organisms. This is synthesized by
the enzyme adenylate cyclase and its concentration is related to
glucose level.
 In E. coli a protein called cAMP receptor protein catabolite activator
protein (CRR) and cAMP bind together to form a complex (cAMP-CRP)
that is a regulatory element for the lac system. The cAMP-CRP complex
must be bound to a specific base sequence (activation site) in the
promoter region in order for transcription of the lac operon to occur.
Thus the cAMP–CRP complex is a positive regulator i.e. in contrast to
the repressor, a negative regulator. The lac operon is therefore
regulated both positively and negatively
Cis- and trans-acting factors
 In both prokaryotes and eukaryotes, the regulation of transcription
involved DNA sequence and factors which can act in two ways;
 direct physical association with the gene undergoing regulation -- cis-
regulation
 Indirect – first producing a diffusible protein product that recognizes a
target gene or genes – trans-regulation.
 Cis – regulation involves promoters and closely associated DNA
regions such as operators which exert this effect as a consequence of
their DNA sequences being associated with the DNA of the gene.
However, the genes for tans-acting factors do not need to be in close
proximity with the target genes. In eukaryotes, the genes for the trans-
acting factors are often located on a different chromosome from the
target gene.
 Mutations which result in an inability to transcribe a gene can be cis- or
trans – acting. e.g. cis defect in lac P of the lac operon prevents the
binding on RNA polymerase to this DNA regions thereby preventing
transcription of the gene. By contrast in trans- acting lac I mutation the
gene product i.e. repressor protein is altered in such a way that it is no
longer able the gene preventing transcription.