Download Biochemistry

The First Page of Teaching Plan
No.
course
Biochemistry
specialty
clinic
class
2015-2
teacher
Chen yan
period
3h
students’
level
undergraduate
professional
title
Biochemistry associate professor
time of
writing
2016.12
chapter
Regulation of gene expression
time of
using
2016-2017(1)
objectives
and
requirements
keys and
difficulties
updated
information
arrangement
teaching
methods
books and
references
teachers’
group
discussion
about the
plan
1.Master the concept of regulation of gene expression and the regulation of transcription in
prokaryotes.
2.Familiar with the characteristics of regulation of transcription in prokaryotes, the structure
of genome in eukaryotes, the specificity and features of gene expression.
3.Understand the regulation of transcription gene expression in eukaryotes at the level of
transcription.
Keys: Concept of gene expression regulation; Prokaryotic gene expression regulation
Difficulties: lac operon and its mechanism in prokaryotes; cis-acting element and
trans-acting factor
no
review the content last class(5min); the definition of gene regulation (10min); Principles of
Gene Regulation(30min); operon (15min); lac operon and its mechanism (50min); cis-acting
element and trans-acting factor (35min); discuss and summarize(5min).
Using CAI to explain, enlightening method
Lippincott’s illustrated review :Biochemistry Pamela C. Champe
wilkins 2009
Biochemistry
the second edition
High Education Press 2002
author: Reginald H. Garrett, Charles M. Grisham
According to learn the principles of gene regulation, then to understand prokaryotic and
eukaryotic gene expression. The lac operon and its mechanism in prokaryotes should be
lectured clearly.
Agree apply in class.
comments
from the
department
Lippincott’s willam &
Sign name:
（Content）
Lesson plan for page
Chapter 12
Regulation of Gene Expression
I.Teaching Goals
It is based on a mastery of basic concepts and principles of regulation and control of
gene expression, then study the regulation of transcription in prokaryotes and to
know(understand) the regulation of transcription gene expression in eukaryotes at the
level of transcription.
II.Teaching Demands
1.Master the concept of regulation of gene expression and the regulation of
transcription in prokaryotes.
2.Familiar with the characteristics of regulation of transcription in prokaryotes, the
structure of genome in eukaryotes, the specificity and features of gene expression.
3.Understand the regulation of transcription gene expression in eukaryotes at the
level of transcription.
III.Teaching Contents
1. The basic concepts and principles of regulation and control of gene expression
The concept of gene expression, temporal and spatial specificity and the features
of gene expression it includes constitutive gene expression, induction and repression.
2. The basic principle in regulation of gene expression
The regulation of gene expression in multi-levels and basic factor of gene
transcription activation.
3. The regulation of gene expression in prokaryotes
The feature in regulation of prokaryotic gene transcription. The mechanism of
gene transcription at initiation regulated by lac operon in E.coil.
4. The regulation of gene expression in eukaryotes
The feature of eukaryotic genome structure, the concept of cis-acting element and
trans-acting factor. Study the feature of eukaryotic gene expression and transcription
regulation of RNA polⅡat initiation by self.
IV. Teaching period
3h
Lesson plan for page
（Content）
Chapter 12 Regulation of Gene Expression
Organisms adapt to environmental changes by altering gene expression. In order for
the organism to adapt to its environment and to conserve energy and nutrients, the
expression of genetic information must be cued to extrinsic signals and respond only
when necessary.
Mammalian cells possess about 1000 times more genetic information than does the
bacterium Escherichia coli. Much of this additional genetic information is probably
involved in regulation of gene expression during the differentiation of tissues and
biologic processes in the multicellular organism and in ensuring that the organism
can respond to complex environmental challenges.
Control of transcription ultimately results from changes in the interaction of
specific binding regulatory proteins with various regions of DNA in the controlled
gene. This can have a positive or negative effect on transcription. Transcription
control can result in tissue-specific gene expression, and gene regulation is
influenced by hormones, heavy metals, and chemicals. In addition to transcription
level controls, gene expression can also be modulated by gene amplification, gene
rearrangement, posttranscriptional modifications, and RNA stabilization.
Analysis of the regulation of gene expression in prokaryotic cells helped establish
the principle that information flows from the gene to a messenger RNA to a specific
protein molecule. These studies were aided by the advanced genetic analyses that
could be performed in prokaryotic and lower eukaryotic organisms. In this chapter,
the initial discussion will center on prokaryotic systems.
1.the definition of gene regulation
Regulation of gene expression (gene regulation) refers to the cellular control of the
amount and timing of changes to the appearance of the functional product of a gene.
Although a functional gene product may be an RNA or a protein, the majority of
the known mechanisms regulate the expression of protein coding genes. Any step of
the gene's expression may be modulated, from DNA-RNA transcription to the
post-translational modification of a protein. Gene regulation gives the cell control
over its structure and function, and is the basis for cellular differentiation,
morphogenesis and the versatility and adaptability of any organism.
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2.Principles of Gene Regulation
2.1 the definition of gene expression
Genes are functional units of DNA that contain the instructions for making
proteins or RNA. And it is the basic unit in inheritance.
A genome is the complete collection of hereditary information for an individual
organism. Homo sapiens, for example, have 22 autosomes plus an X chromosome or
Y chromosome.
Gene expression is the process by which a gene's DNA sequence is converted into
the functional proteins of the cell.
2.2 The specificity of gene expression
2.2.1 Temporal specificity
Requirements for some gene products change over time. The need for enzymes in
certain metabolic pathways may wax and wane as food sources change or are
depleted.
This protein is alpha-fetoprotein, a major plasma protein produced by the yolk sac
and the liver during fetal life. Expression of AFP (Alpha-fetoglobulin) in adult cells
is low, however synthesis aberrantly occurs in adult liver cancer cells.
Alpha-fetoprotein expression in adults is often associated with hepatoma or teratoma.
However, hereditary persistance of alpha-fetoprotein may also be found in
individuals with no obvious pathology. The protein is thought to be the fetal
counterpart of serum albumin, and the alpha-fetoprotein and albumin genes are
present in tandem in the same transcriptional orientation on chromosome 4.
2.2.2 Spatial specificity (Tissue-specific regulation)
Specialization of cellular function can dramatically affect the need for various
gene products; an example is the uniquely high concentration of a single
protein—hemoglobin—in erythrocytes. Given the high cost of protein synthesis,
regulation of gene expression is essential to making optimal use of available energy.
Adult tissue-specific stem cells have the capacity to self-renew and generate
functional differentiated cells that replenish lost cells throughout an organism’s
lifetime.
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2.3 Features of Gene Regulation
2.3.1 housekeeping gene
The expression of some genes is constitutive, essential for basic processes
involving in cell replication and growth. It means that they are expressed at a
reasonably constant rate and not known to be subject to regulation. Genes for
products that are required at all times, such as those for the enzymes of central
metabolic pathways, are expressed at a more or less constant level in virtually every
cell of a species or organism. These are often referred to as housekeeping genes.
Unvarying expression of a gene is called constitutive gene expression.
2.3.2 inducible gene
An inducible gene is one whose expression increases in response to an inducer or
activator, a specific positive regulatory signal. In general, inducible genes have
relatively low basal rates of transcription. By contrast, genes with high basal rates of
transcription are often subject to down-regulation by repressors.
2.4 Features of Regulation
Inducer - The substance which induces gene expression or protein synthesis is
called as inducer. The phenomenon is called as induction.
Repressor - The substance which stops or represses the expression of specific
genes is called as repressor and the phenomenon is called as repression.
If the presence of a specific regulatory element enhances the level of gene
expression, then that is called as positive regulation and the molecule is called as
activator, e.g. lac operon If the presence of a specific regulatory molecule will reduce
the gene expression, then it is called as negative regulation and the molecule is called
as repressor.
Jacob and Monad in 1961 proposed a model which explains how a bacteria
metabolizes lactose and tryptone, and the model is called as Jacob-Monad model.
Bacteria use a positive control to metabolize lactose and negative control or
regulation to metabolize tryptone.
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2.5. Regulated stages of gene expression(eurkaryote)
Any step of gene expression may be modulated, from the DNA-RNA transcription
step to post-translational modification of a protein. Following is a list of stages where
gene expression is regulated:
Chemical and structural modification of DNA or chromatin;
Transcription;
Translation;
Post-transcriptional modification;
RNA transport;
mRNA degradation;
Post-translational modifications;
3.Gene Control in Prokaryotes
In bacteria, genes are clustered into operons: gene clusters that encode the
proteins necessary to perform coordinated function, such as biosynthesis of a given
amino acid. RNA that is transcribed from prokaryotic operons is polycistronic a term
implying that multiple proteins are encoded in a single transcript.
--control of gene expression enablesindividual bacteria to adjust theirmetabolism to
environmental change
--cells vary amount of specific enzymesby regulating gene transcription
-turn gene on or turn gene off
ex. if you have enough tryptophanin your cell then you don’t need to make
enzymesused to build tryptophan
-waste of energy
-turn off gene which codes for enzyme
The activity of RNA polymerase at a given promoter is in turn regulated by
interaction with accessory proteins, which affect its ability to recognize start sites.
These regulatory proteins can act both positively (activators) and negatively
(repressors). The accessibility of promoter regions of prokaryotic DNA is in many
cases regulated by the interaction of proteins with sequences termed operators. The
operator region is adjacent to the promoter elements in most operons and in most
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cases the sequences of the operator bind a repressor protein. However, there are
several operons in E. coli that contain overlapping sequence elements, one that binds
a repressor and one that binds an activator.
As indicated above, prokaryotic genes that encode the proteins necessary to
perform coordinated function are clustered into operons. Two major modes of
transcriptional regulation function in bacteria (E. coli) to control the expression of
operons. Both mechanisms involve repressor proteins. One mode of regulation is
exerted upon operons that produce gene products necessary for the utilization of
energy; these are catabolite-regulated operons. The other mode regulates operons that
produce gene products necessary for the synthesis of small biomolecules such as
amino acids. Expression from the latter class of operons is attenuated by sequences
within the transcribed RNA.
A classic example of a catabolite-regulated operon is the lac operon, responsible
for obtaining energy from β-galactosides such as lactose. A classic example of an
attenuated operon is the trp operon, responsible for the biosynthesis of tryptophan.
Operon - An operon is a set of genes which are linked and are under the control of
one promoter or operator. These genes accomplish one single task. An operon
basically consists of two categories of genes.
1.Structural genes - These genes are segments of DNA, which code for
functional peptides, or enzymes or proteins. Proteins of structural genes directly
interact with the inducer or accomplish a single task.
2.Control genes - These are genes which are primarily responsible for
controlling the structural genes by producing an inducer or repressor substance.
There are basically three types of genes.
i. Regulator genes - The regulator gene (R) produces some specific enzymes
which act as repressor substances. This repressor binds to the operator gene and thus
stops the expression of structural genes.
ii. Promoter gene - The promoter gene (P) is the DNA segment at which RNA
polymerase binds. It initiates the transcription of the structural genes.
iii. Operator gene -The operator gene (O) is the segment of DNA which exercises
a control over transcription. It lies close to the structural gene and the repressor binds
to it.
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Lactose Operon in E coli –A inducible operon
In an inducible operon, the repressor actively blocks the gene from transcription.
The controller molecule attaches to the repressor removing it from the operator and
transcription proceeds.
1.Structure and catabolite of glucose
The molecular mechanisms responsible for the regulation of the genes involved in
the metabolism of lactose are now among the best-understood in any organism.
β-Galactosidase hydrolyzes the β-galactoside lactose to galactose and glucose.
The structural gene for β-galactosidase (lacZ) is clustered with the genes
responsible for the permeation of galactose into the cell (lacY) and for
thiogalactoside transacetylase (lacA). The structural genes for these three enzymes,
along with the lac promoter and lac operator (a regulatory region), are physically
associated to constitute the lac operon. This genetic arrangement of the structural
genes and their regulatory genes allows for coordinate expression of the three
enzymes concerned with lactose metabolism.
When E coli is presented with lactose or some specific lactose analogs under
appropriate nonrepressing conditions (eg, high concentrations of lactose, no or very
low glucose in media; see below), the expression of the activities of β-galactosidase,
galactoside permease, and thiogalactoside transacetylase is increased 100-fold to
1000-fold. Upon removal of the signal, ie, the inducer, the synthesis of these three
enzymes declines. When E coli is exposed to both lactose and glucose as sources of
carbon, the organisms first metabolize the glucose and then temporarily stop growing
until the genes of the lac become induced to provide the ability to metabolize lactose
as a usable energy source. Although lactose is present from the beginning of the
bacterial growth phase, the cell does not induce those enzymes necessary for
catabolism of lactose until the glucose has been exhausted. This phenomenon was
first thought to be attributable to repression of the lac operon by some catabolite of
glucose; hence, it was termed catabolite repression. It is now known that catabolite
repression is in fact mediated by a catabolite gene activator protein (CAP) in
conjunction with cAMP. This protein is also referred to as the cAMP regulatory
protein (CRP). The expression of many inducible enzyme systems or operons in E
coli and other prokaryotes is sensitive to catabolite repression, as discussed below.
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2. regulation
(1)negative regulation
The physiology of induction of the lac operon is well understood at the molecular
level.Expression of the normal lacI gene of the lac operon is constitutive; it is
expressed at a constant rate, resulting in formation of the subunits of the lac
repressor. Four identical subunits with molecular weights of 38,000 assemble into a
lac repressor molecule. The LacI repressor protein molecule, the product of lacI, has
a high affinity for the operator locus. The operator locus is between the promoter site,
at which the DNA-dependent RNA polymerase attaches to commence transcription,
and the transcription initiation site of the lacZ gene.
When attached to the operator locus, the LacI repressor molecule prevents
transcription of the operator locus as well as of the distal structural genes, lacZ, lacY,
and lacA. Thus, the LacI repressor molecule is a negative regulator; in its presence
(and in the absence of inducer), expression from the lacZ, lacY, and lacA genes is
prevented. A lactose analog that is capable of inducing the lac operon while not itself
serving as a substrate for β-galactosidase is an example of a gratuitous inducer. An
example is isopropylthiogalactoside (IPTG).
The addition of lactose or of a gratuitous inducer such as IPTG to bacteria growing
on a poorly utilized carbon source(such as succinate) results in prompt induction of
the lac operon enzymes. Small amounts of the gratuitous inducer or of lactose are
able to enter the cell even in the absence of permease. The LacI repressor
molecules—both those attached to the operator loci and those free in the
cytosol—have a high affinity for the inducer. Binding of the inducer to a repressor
molecule attached to the operator locus induces a conformational change in the
structure of the repressor and causes it to dissociate from the DNA because its
affinity for the operator is now 103 times lower than that of LacI in the absence of
IPTG. If DNA-dependent RNA polymerase has already attached to the coding strand
at the promoter site, transcription will begin. Derepression of the lac operon allows
the cell to synthesize the enzymes necessary to catabolize lactose as an energy
source.
Based on the physiology just described, IPTG-induced expression of transfected
plasmids bearing the lac operator-promoter ligated to appropriate bioengineered
constructs is commonly used to express mammalian recombinant proteins in E coli.
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(2)positive regulation
In order for the RNA polymerase to efficiently form a PIC at the promoter site,
there must also be present the catabolite gene activator protein (CAP) to which
cAMP is bound.
By an independent mechanism, the bacterium accumulates cAMP only when it is
starved for a source of carbon. In the presence of glucose—or of glycerol in
concentrations sufficient for growth—the bacteria will lack sufficient cAMP to bind
to CAP because the glucose inhibits adenylyl cyclase, the enzyme that converts ATP
to cAMP . Thus, in the presence of glucose or glycerol, cAMP-saturated CAP is
lacking, so that the DNA-dependent RNA polymerase cannot initiate transcription of
the lac operon. In the presence of the CAP-cAMP complex, which binds to DNA just
upstream of the promoter site, transcription then occurs, and stimulates RNA
transcription 50-fold.
Studies indicate that a region of CAP contacts the RNA polymerase α subunit and
facilitates binding of this enzyme to the promoter. Thus, the CAP-cAMP regulator is
acting as a positive regulator because its presence is required for gene expression.
The lac operon is therefore controlled by two distinct, ligand-modulated DNA
binding trans factors; one that acts positively (cAMP-CRP complex) and one that acts
negatively (LacI repressor). Maximal activity of the lac operon occurs when glucose
levels are low (high cAMP with CAP activation) and lactose is present (LacI is
prevented from binding to the operator).When the lacI gene has been mutated so that
its product, LacI, is not capable of binding to operator DNA, the organism will
exhibit constitutive expression of the lac operon. In a contrary manner, an organism
with a lacI gene mutation that produces a LacI protein which prevents the binding of
an inducer to the repressor will remain repressed even in the presence of the inducer
molecule, because the inducer cannot bind to the repressor on the operator locus in
order to derepress the operon. Similarly, bacteria harboring mutations in their lac
operator locus such that the operator sequence will not bind a normal repressor
molecule constitutively express the lac operon genes.
Mechanisms of positive and negative regulation comparable to those described
here for the lac system have been observed in eukaryotic cells.
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4.Gene expression in eukaryotic cells
--the control mechanisms of gene expression are varied and complex
--cellular differentiation of eukaryotic cells depends on turning on and off genes in
the proper sequence
(1)DNA packing in eukaryotic chromosomes helps regulate gene expression
--DNA wrapping around histonesand other proteins into nucleosomes, coiling,
supercoiling, and additional folding into chromosomes
--DNA packing prevents gene expression, most likely by preventing transcription
(2)Initiation of transcription -Complex assemblies of proteins control eukaryotic
transcription
--Transcription factors interact with enhancer sites (a DNA sequence) in
regulating the binding of RNA polymerase to a gene’s promoter
--The binding of activators to enhancers initiates transcription
--Repressor proteins interact with nucleotide sequences called silencers
-inhibit the start of transcription
Like the prokaryotic gene, the eukaryotic gene has a number of regions, each
important to transcription. The components of the eukaryotic transcription complex
include:
CIS Acting Elements (Control elements)
A specific promoter region within the control elements, which indicates the
starting point for transcription.
Eukaryotic genes are regulated by promoter elements located just upstream (5 ')
from the transcription initiation sites in a manner quite similar to the regulation of
prokaryotic genes. In addition to the nearby promoters, many eukaryotic genes are
also regulated by more distant cis acting elements called enhancers and silencers.
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A region called the enhancer that stimulates the binding of RNA polymerase to
the promoter region. The enhancer region is comprised of non-coding DNA that
binds to transcription factors called activators. Activators fold the DNA so that the
enhancers are brought to the promoter region of the gene where they bind to
additional transcription factors.
Silencers are control elements that can inhibit transcription. A transcription factor
that binds to a silencer control element and blocks transcription is called a repressor.
Specific Transcription Factors
Eukaryotic transcription begins with the formation of a pre initiation complex
formed by the amalgamation of a group of general transcription factors. Proteins that
exert control over transcription at specific promoters are the specific transcription
factors.
These proteins generally have two domains, a domain that recognizes a specific
DNA sequence and a domain that recognizes another protein like the pre initiation
complex. A majority of specific transcription factors act by recruiting the components
of the RNA polymerase holoenzyme.
(3)Eukaryotic RNA may be spliced in more than one way
Introns have been shown to function in gene regulation
--alternative RNA splicing
--increasing the possibility of crossovers between exons
--increasing genetic diversity
(4)Regulation in the cytoplasm
mRNA breakdown: long-lived mRNAs get translated into many protein molecules
than do short-lived ones
translation
Protein alterations: Post-translational control mechanisms in eukaryotes often
involve cutting polypeptides into smaller, active final products
Protein breakdown