11/30/2015

Fundamental Genetics
Lecture 14

Regulation of
Gene Expression

Genetic Regulation in Prokaryotes

The E. coli genome can code up to 4000 polypeptides
But different polypeptides have different number of copies
Some proteins are 5-10 copies while others could be as much as
100,000 copies per cell.
What regulates gene expression? How it is regulated?
Regulation is influenced by substances from the environment
Example: Regulation of enzymes involved in the breakdown of lactose

John Donnie A. Ramos, Ph.D.

Department of Biological Sciences
College of Science
University of Santo Tomas

When E. coli are cultured in medium without lactose, enzymes needed

for lactose breakdown are not synthesized but the enzymes are
produced at a great number when lactose is added to medium.
Operon group of related genes (clustered together in a
chromosome) that are involved in the regulation of gene expression

Genetic Regulation in Prokaryotes

Inducible synthesis production of proteins resulting from the presence of

inducer molecule in the environment.

Example of inducer molecule: lactose (influences the synthesis of -galactosidase,

permease, and transacetylase)

Constitutive synthesis continuous production of proteins regardless of the

presence or absence of molecules in the environment

Repressible synthesis - production of proteins are inhibited in the presence of

a repressible molecule in the environment.

The Lactose (Lac) Operon

Housekeeping genes ribosomal proteins

Trans-acting
element

Cis-acting
element

Regulation may be under:

Negative control gene expression occurs unless shut off by a regulator molecule

Positive control gene expression occurs only if stimulated by a regulatory

molecule

Transacetylase

Polycistronic
mRNA

The Lactose (Lac) Operon

Lactose or its analog are
inducer molecules

Permease (entry
of lactose into cell)

(removes toxic
products of
lactose
digestion

Enzymes involved in the synthesis of tryptophan are not produced when

tryptophan is present in the culture medium

-Galactosidase

The Lactose (Lac) Operon

Negative control

Translation
occurs only when
regulator
molecules are
present

Inducible operon

Lactose as
inducer molecule

Repressor
molecule is
allosteric
IPTG
(Isopropylthiogalactosidase)
lactose analog

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Mutant Genes in the Lac Operon

Mutant Genes in the Lac Operon

Mutation on lactose binding site

CAP Protein

Tryptophan (Trp) Operon

Positive control
of the Lac
operon
Catabolite
repression
influenced by
glucose

Structural genes

Chorismate

Anthranilate

Anthranilate
synthase

P-ribosyl
transferase

Phosphoribosyl

Tryptophan Synthesis

Tryptophan (Trp) Operon

Repressible operon
repressor molecule inhibits
transcription
When tryptophan is present
in medium, enzymes are not
produced
Negative control
Tryptophan is a co-repressor
Attenuation termination of
transcription due to the
presence of attenuator gene
(regulation at the RNA level
by forming RNA loops

Indole glycerol
phosphate
synthase

Carboxyphenylamin
ol-1-deoxyribulase5-phosphate
(CDRP)

Tryptophan
synthetase

Indole glycerol
phosphate

Tryptophan

Genetic Regulation in Eukaryotes

Eukaryotic genes need to be expressed
at the right cell type, right time, and
right amount
Contains mechanisms for the
activation or repression of a given
gene
More complex that prokaryotes
Gene expression can be regulated at
different levels

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Promoters

Enhancers
Interacts with trans-acting regulatory proteins
Characteristics of enhancers:
No fix position in a gene (before, within or after a gene)
They can operate away from a given gene (as much as 50 kb)
Sequence of enhancer could be inverted
Not specific regulation (can positively regulate any gene near to it)

Recognition sites for RNA polymerase

Initiate transcription

Examples:
Immunoglobulin heavy chain gene enhancer is located in an
intron
globin gene downstream enhancer

Controls the transcription of a given gene with the formation of

loops

Transcription Factors
Control where, when and what rate genes are expressed.
A complex of different domains and subunits
Domains:

DNA-binding domain binds to promoter or enhancer regions

Trans-acting domain binds other subunits of TF to polymerases

Structural Motifs of TFs

Helix-turn-helix motif
No specific sequence involved but
geometric conformation
Motif cannot function alone (part of DNA
binding domain
Found in a homeobox encoded by a
homeodomain (coding 60 aa, mostly basic
aa arg and lys)

Zinc finger motif

Cluster of 2 cys and 2 his covalently linked

to zinc atom
Consists of 23 aa with 7-8 aa forming loop

Basic leucine zipper motif

Assembly of Transcription Complex

TFIID also called TATA-binding protein (TBP)
Composed of 11 TBP-associated factors (TAFs)
Involved at least 20 bp DNA

Other transcription factors interact with each

other by protein-protein interactions
TFIIH with helicase activity
Rate of transcription increases when complex
binds to a group of TF bound to enhancer
element

35 aa with repeating 4 Leu spaced 7 aa

apart
Forms dimers (interaction between Leu)

Chromatin Remodeling
Alteration of the chromatin
assembly (separation of DNA
from histone proteins)
In order for transcription factors
and polymerase to gain access
to a DNA molecules for
transcription
Acetylation of histones
decreases interaction with DNA
CBP coactivator contains
acetyltransferase activity.

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DNA Methylation
Addition or removal of methyl groups to DNA bases
Occurs immediately after replication
Often involves cytosine (5 % of all cytosines are methylated)
Addition of methyl group occurs at the 5th Carbon of
pyrimidine ring protrudes to major groove
Presence of methyl group affects the binding of transcription
factors

Gene Regulation by Steroid Hormone

Hormone receptor proteins
contains two zinc fingers
that can bind with DNA
sequences
Hormone responsive
elements (HREs) DNA
sequences where hormone
receptors bind

Occurs in a DNA sequence called CpG motifs

Higher methylation of a DNA sequence is related low gene
expression or even inactivation

Example: Barr body (inactivated X chromosome) highly

methylated

Posttranscriptional Regulation

RNA splicing
Addition of 5 cap
Formation of poly A tail (3 end)
Complex formation with proteins
Alternative Splicing (generates different forms of proteins isoforms)

PPT-preprotachynin
(neuropeptides)