gene expression in prokaryotes, lac operon, tryptophan operon

1. GENE EXPRESSION IN
PROKARYOTES
Mrs. Praveen Garg
VITS College, Satna

2. INTRODUCTION
• Prokaryotic gene expression (both transcription and translation)
occurs within the cytoplasm of a cell due to the lack of a defined
nucleus, and the DNA is freely located within the cytoplasm.
• Gene expression in prokaryotes occurs primarily at the level of
transcription.
• Bacteria genes that encode for proteins are closely related with cis-
acting regulatory elements that determine the transcription of these
genes, thus these genes are regulated in a coordinated way. These
clusters of genes are called operons, and their ranscription product is a
single polycistronic mRNA.
• Organization of genes in operons contributes to the regulation of gene
expression.
• The operon can therefore be categorized as inducible or repressible.

3. Inducible operon
• The include genes that encode for enzymes that take part in
metabolic pathways as the expression of the gene is controlled by
the substrate
• Example: the "Lac Operon"
Repressible operon
• The include genes that encode for enzymes involved in
biosynthetic pathways, and the expression of the gene is
controlled by the end-product of the pathway
• Example: the "Trp Operon"

4. • It was Jacob and Monod in 1961 who proposed the operon
model for the regulation of transcription.
• The operon model proposes three elements:
• A set of structural genes (i.e. genes encoding the proteins to be
regulated);
• An operator gene, which is a DNA sequence that regulates
transcription of the structural genes.
• A regulator gene which encodes a protein that recognizes the
operator sequence.

5. OPERATORS
• These are segments of DNA that regulate the activity of the
structural genes of the operon.
• If the operator is not bound by a repressor molecule, then the
RNA polymerase can pass over the operator and move to the
protein coding genes.
• If the operator, on the other hand, is bound by a repressor
molecule , then the RNA polymerase is blocked behind the
repressor molecule.
• However, when an "Inducer molecule" is present, it binds the
repressor molecule causing it to change shape, rendering it
incapable of binding the operator. The RNA polymerase moves
freely.

6. The lactose operon (lac operon)
• The lactose operon contains 3 structure genes, lac Z, lacY and
lacA.
• It contain promoter and operator gene.
• These genes encode for enzymes required to metabolize lactose-
beta-galactosidase, lactose permease, and beta-galactoside
transacetylase.
• Another regulatory gene, Lac I, is expressed separately and lies
upstream of the operon. This gene ecodes for the lac repressor
which regulates the expression of Lac Z, Y ans A.
• In lac operon, lactose use as a substrate or inducer for regulation
of genes.

8. • In the absence of lactose:
• The lac repressor bind to a DNA sequence called the "operator"
(found between the lac Z gene and the lac promoter).
• In this way the lac repressor blocks the path of RNA polymerase
to reach the lac Z,Y and A genes, operon remains switched off.
• In the presence of lactose:
• Lactose molecules are metabolized by the lac enzymes, an
intermediate is formed called allolactose (an isomer of lactose)
• Allolactose acts as an inducer by binding to the lactose repressor
and changing its conformation - therefore it can no longer bind
to the operator.

11. • In the presence of glucose (even if present together with
lactose):
• Glucose is preferred because it requires less energy to be broken down.
• The operon senses the glucose presence and by a mechanism called
"catabolite repression" it is switched off.
• The regulatory protein "CAP" (Catabolite Activator Protein) binds to a
DNA sequence upstream to the lac promoter and enhances binding of
the RNA polymerase leading to an increased transcription of the
operon.
• However, CAP will only bind behind the promoter only if cAMP is
bound on it. Adenylate cyclase though, the enzyme required for the
synthesis of cAMP, is inhibited by glucose and the operon will be
eventually expressed at very low rate.

12. If glucose is present in mediuum

13. The Tryptophan operon (Trp operon)
• The trp operon is an operon—a group of genes that is used, or
transcribed, together—that codes for the components for production
of tryptophan.
• It includes 5 genes, trpE, trpD, trpC, trpB, and trpA, involved in
Tryptophan synthesis.
• The genes are expressed as a single mRNA strand, transcribed from an
upstream promoter.
• Another regulatory gene encodes for a trp repressor.
• There is also the trp operator that is found just downstream from the
promoter.
• If tryptophan is present it will bind the trp repressor. This enables it to
bind the operator and block the RNA polymerase. This is a model of end-
product inhibition, since tryptophan is the end product of tryptophan
biosynthesis.
• Also unlike the lac operon, the trp operon contains a leader peptide and
an attenuator sequence which allows for graded regulation.

16. If tryptophan is absent:
• In this case, inactive repressor can not bound with operator.
Thus RNA polymerase move in to structural gene, and
synthesize polypeptides.
If tryptophan is present:
• In this case, inactive repressor bind with tryptophan present in
medium and change in to active state.
• This active repressor bound with operator and block the RNA
polymerase to moving forward, and terminate the
transcription process.

19. Attenuation
• Attenuation is a mechanism for reducing expression of the trp
operon when levels of tryptophan are high. However, rather than
blocking initiation of transcription, attenuation prevents completion of
transcription.
• The 5′ end of the polycistronic mRNA transcribed from the trp operon
has a leader sequence upstream of the coding region of the trpE
structural gene.
• This leader sequence encodes a 14 amino acid leader peptide containing
two tryptophan residues.
• The leader sequence contains four regions (numbered 1–4) that can
form a variety of base paired stem-loop (‘hairpin’) secondary structures.
• If region 3 and region 4 base pair with each other, they form a loop like
structure called attenuator and it function as transcriptional
termination. If pairing occur between region 3 and region 2, then no
such attenuator form so that transcription continues.
• This loop like structure called attenuator and it function as
transcriptional termination.

20. When Tryptophan is abundant
• When tryptophan is abundant, ribosomes bind to the trp polycistronic
mRNA that is being transcribed and begin to translate the leader sequence.
• The leader sequence can have base pairing and form hair pin loop like
structure, cause termination.
• Therefore, when tryptophan is present, further transcription of the trp
operon is prevented.

21. When Tryptophan is scarce
• If, however, tryptophan is in short supply, the ribosome will pause at the
two trp codons contained within sequence 1.
• This leaves sequence 2 free to base pair with sequence 3 to form a 2:3
structure (also called the anti-terminator), so the 3:4 structure cannot
form and transcription continues to the end of the trp operon.

22. • Hence the availability of tryptophan controls whether
transcription of this operon will stop early (attenuation) or
continue to synthesize a complete polycistronic mRNA.
• Overall, for the trp operon, repression via the trp repressor
determines whether transcription will occur or not and
attenuation then fine tunes transcription.

23. THANK YOU