Transcript Genetics - PCB 3063

Genetics - PCB 3063
• Today’s focus:
– Gene Regulation
– We will focus on two major questions
today:
– How do regulatory genes control the activity of
multiple genes?
– How does gene regulation differ between
prokaryotes and eukaryotes?
Homework - Due Next Tuesday
• The E. coli gal operon is regulated by a repressor (galR) that
binds to the galO operator to regulate the structural genes galE,
galT, and galK. Expression is induced by the presence of
galactose in the medium. If you had cells of the following types,
would you expect the structural genes to be inducible,
constitutive, or not expressed (give each structural gene) -–
–
–
–
galR- galO+ galE+ galT+ galK+
galR+ galOc galE+ galT+ galK+
galR- galO+ galE+ galT+ galK+ / galR+ galO+ galE- galT+ galK+
galR- galOc galE+ galT+ galK- / galR+ galO+ galE- galT+ galK+
• Note: the last two are merodiploids.
• Which eukaryotic RNA polymerase transcribes the following
genes -– tRNAs
– mRNAs
– rRNAs
First, Let’s Finish Introns...
• Typical (canonical) introns follow the GT-AG rule.
• Plants and animals also have a small number of non-canonical
introns that do not follow the GT-AG (or GU-AG) rule.
• There are also tRNA introns removed by specific enzymes.
• Self-splicing introns that directly catalyze their own removal have
also been found in various genomes.
• rRNA is also processed by removal of external and internal
transcribed spacers.
– However, there is no splicing after ETS and ITS removal, so the
ITS and ETS are not introns.
RNA Processing - Alternative Splicing
• The process of splicing can be controlled - there are
introns that are spliced only under specific conditions.
• For example, sex determination in Drosophila results
from a set of genes with alternatively spliced introns.
– E.g., note the skipping of intron 3 in the Sxl (sex lethal) gene of
female Drosophila.
“Inside-Out” Genes and Inteins
• In most genes, the introns are removed and degraded
while the EXON sequences are functional.
• The snoRNAs are involved in ribosome assembley
(small nucleolar RNAs).
– One snoRNA (called U22) is present in an intron and the exons
are exported to the cytoplasm to be degraded - this is an
“inside-out” gene
• Tycowski, K. T., Shu, M.-D., and Steitz, J.A. (1996) Nature 379:464-466.
• Splicing of proteins has also been described.
– These protein introns are called INTEINS.
– Inteins are autocatalytically removed from the spliced EXTEIN
sequences.
– Surprisingly, inteins are related to nucleases.
– More information on inteins can be found at:
• http://www.neb.com/inteins/intein_intro.html
Gene Regulation
• There are multiple points at which gene expression is
controlled. These include:
– Transcription
– Translation
– Degradation (RNA
and/or protein)
– Control of protein
activity (e.g., by
phosphorylation)
Nucleus
DNA
1¡ t ransc ript (hnRNA)
DNA
degradat ion
degradat ion
mRNA
mRNA
Protein
Prokaryotic Gene Regulation
mRNA
Protein
Eukaryotic Gene Regulation
degradat ion
degradat ion
• Eukaryotes can also control the processing of mRNA
and the export of mRNA from the nucleus.
– But transcription and translation can interact in prokaryotes.
Terminology
• A gene that shows increased expression under certain
circumstances is said to be INDUCIBLE.
– The observation that a gene is induced under certain
circumstances does not establish the type of control.
– For this reason, one typically discusses changes in the
accumulation of mRNAs or proteins.
• E.g, an increased amount of mRNA can reflect either transcription
(increased), degradation (decreased), or both phenomena.
• However, regulation at the level of transcription is fairly common.
• Many genes are expressed under a large number of
conditions (and in many tissues in complex organisms).
These genes are usually called housekeeping genes.
– Housekeeping genes are expressed CONSTITUTIVELY.
– Karström (1859) concluded that bacterial enzymes may be classified as either
constitutive or adaptive. The former are invariably present…the presence of the
latter depends on the presence of the substrate upon which they act.
The lac Operon
• The lac operon is a paradigm for gene regulation.
• Regulation of the lac operon occurs at the level of
transcription.
• Study of the lac operon are based upon a simple
observation - E. coli cells synthesize the enzyme
b-galactosidase only when two conditions are met:
– Lactose is present.
– Glucose is absent.
A
B
1 2 50
1 0 00
Graph of b-galactosidase
activity from cells grown
in medium with lactose &
glucose.
A. Glucose exhausted.
B. Glucose added back.
750
b- galacto sidase
act iv it y
500
250
0
0
1
2
3
Time (h)
4
5
b-galactosidase
•
b-galactosidase catalyzes the cleavage of lactose into
glucose and galactose.
Glucose
+
H2O
Galactose
Lactose
• In addition to b-galactosidase, both a lactose permease
and b-galactoside transacetylase are induced.
• b-galactosidase converts lactose to ALLOLACTOSE,
necessary for induction:
Allolactose
(b-1,6 linkage)
The lac Operon - Genes
• There are four major genes involved in the lac operon:
–
–
–
–
lacZ - encodes b-galactosidase
lacY - encodes b-galactoside permease
lacA - encodes b-galactoside transacetylase
lacI - encodes the lac repressor
• In addition, one must consider RNAP and CRP (cyclic
AMP receptor protein) and several cis acting sites.
Trans-Acting Factors & Cis-Acting Sites
• There are two major types of transcriptional regulatory
entities you should consider:
– Trans-Acting Factors - proteins that turn the transcription of
genes on or off.
– Since they are proteins, they are able to diffuse throughout the
cell (hence, they can act in trans - e.g., when they are present
in a merozygote)
– Cis-Acting Sites - these are typically the binding sites for
trans-acting factors.
– Since the are specific sites in the DNA that are necessary for
gene regulation, they can only act when adjacent to the gene
in question.
Trans -Acting Factor
Trans cription
Ci s-Acting Si te
The lac Operon - Repression
• lacI encodes a repressor that binds to the lac operator
(lacO), present between the lac promoter (lacP) and the
structural genes.
– Binding of the lacI gene product to lacO blocks transcription
of the polycistronic mRNA encoded by the lac operon.
– The structural genes of the lac operon will be regulated in a
coordinate fashion, because they are present on the same
mRNA.
The lac Operon - Derepression
• When the lacI encoded repressor binds to inducer
(allolactose) it cannot bind the lac operator (lacO).
– Thus, transcription of the polycistronic lac operon mRNA is
not blocked.
– The structural change in the lac repressor caused by
allolactose reflects a phenomenon called allosteric regulation.
– This model - proposed by F. Jacob & J. Monod - explains
induction by lactose (but not regulation by glucose).
The Operon Concept
• An operon is defined as a unit of co-ordinated gene
activity believed to account for the regulation of
inducible and repressible enzymes in bacteria (and
hence for the regulation of protein synthesis).
– The operon is usually conceived as a linear sequence of
genetic material comprising an operator, a promoter, and one
or more structural genes.
• This terminology was introduced by Jacob and Monod
in the Journal of Molecular Biology (1961)
– Discussing the lac operon they stated that “This genetic unit
of co-ordinate expression we shall call the ‘operon’.”
– The term REGULON is used if there are multiple operons at
distinct locations in the genome that are regulated by the
same trans-acting factors. Operon is used if there is a single
gene or set of genes at one location with this type of control.
Catabolite Repression
• The regulation of the lac operon by glucose reflects
the phenomenon of catabolite repression.
– CRP (cyclic AMP [cAMP] receptor protein), also called CAP
(catabolite activator protein) binds to a cis-acting site and
promotes RNAP binding.
• CRP promotes RNAP binding by bending DNA.
– CRP binds DNA when it binds to cAMP.
• cAMP is produced when glucose is absent.
cAMP and Catabolite Repression
• cyclic AMP (cAMP) accumulates when glucose is absent.
– Adenylcyclase is inhibited by glucose.
– cAMP phosphodiesterase is activated by glucose.
Adenyl
cyclase
cAMP
ATP
Phosphodiesterase
AMP
The lac Operon: Multiple Signals
• These two regulatory systems result in the observed
regulatory pattern: Lactose Glucose cAMP lac Operon
+
+
+
+
-
Low
No mRNA
High Little mRNA
Low
No mRNA
High High mRNA
accumulation
– In addition to these patterns, it was also noted that some
mutations in lac genes abolished the activity of genes that
follow them in the operon.
• e.g., there are lacZ mutants that do not express lacY or lacA.
– These are called POLAR MUTATIONS.
– These mutations are NONSENSE MUTATIONS.
• The phenotype of polar mutations reflects problems with
the translation of downstream cistrons when there are
mutations in the upstream genes.
The trp Operon - Repression
• Tryptophan biosynthetic genes are also regulated, but
the mechanism is quite different:
– In this case, tryptophan (trp) binds to a repressor (the product
of trpR) that works in a manner similar to the lac repressor.
– However, the binding of Trp to the trpR gene product causes it
to bind DNA.
– Thus, Trp is called a COREPRESSOR in this context.
The trp Operon - Attenuation
• However, trp operon regulation is more complex:
– C. Yanofsky established that the leader sequence of the
polycistronic transcript of the trp operon contains a short
open reading frame (ORF) with a ribosome binding site (RBS).
– This ORF has two tryptophan codons, which can cause a
ribosome to stall during translation if it is limiting.
Attenuation - Stem Loops
• The trp operon mRNA leader can adopt several structures:
• If the ribosome stalls, it causes a structure that does not
result in termination.
• Either read-through or the absence of translation allow the
mRNA to adopt a structure that results in termination.
– Could this mechanism of gene regulation occur in eukaryotes?
Attenuation - Variation
• In E. coli and Salmonella, there are other amino acid
biosynthetic operons regulated by attenuation:
Regulatory
Amino
Operon
Leader
Acids
MTRVQFKHHHHHHHPD
His
His
Phe MKHIPFFFAFFFTFP
Phe
Leu MSHIVRFTGLLLLNAFIVR...
Leu
MKRISTTITTTIT ITTGNGAG
Thr
Thr, Ile
MTALLRVISLVVISVVVIII P...
Ilv
Ile, Val
• In B. subtilis, the trp operon mRNA is also controlled
by attenuation.
– However, it does not involve ribosome binding.
– Instead, there is a protein called TRAP (trp RNA binding
attenuation protein) that binds the leader of the trp mRNA.
– TRAP binds Trp (11 molecules) and then binds the leader,
causing attenuation.
GAL Genes:
Eukaryotic Transcriptional Regulation
• Unlike prokaryotes, eukaryotes do not have genes in
operons (most mRNAs are not polycistronic).
• The GAL genes of S. cerevisiae are the paradigm for
eukaryotic gene regulation
• Galactose is metabolized by GAL gene products:
Galactose
Gal1p
UDP-Glu
Gal10p
Gal-1-P
Gal7p
UDP-Gal
Glu-6-P
Glycolysis
Gal5p
Glu-1-P
Eukaryotic
Transcription
• Proteins bind to distal
elements called
ENHANCERS.
• DNA folding allows
these elements to be
far from the start site
for transcription.
• Proteins bound to the
distal sites promote
the binding of RNA
polymerase to the
proximal elements.
Distal
Proximal
GAL Genes: A Transcriptional Program
• The response to galactose is very complex, with a
number of genes being turned on or off.
• The central regulator is a protein called Gal4p.
– Gal4p binds to enhancer elements in DNA and activates
transcription under some circumstances.
Gal4p: A Transcriptional Regulator
• Gal4p binds to enhancer elements near genes that it
regulates (e.g., GAL1).
• Gal4p also binds to Gal80p.
– Gal80p is necessary for activation of gene expression.
• When galactose binds to Gal80p, the Gal4p-Gal80p
complex can activate transcription.
– This activation has now been studied at the level of the whole
genome:
•
This figure shows data from a microarray experiment (Science 290:2306 [2000]).
Examining Transcriptional Regulation
• MICROARRAYS have become very popular as tools to
study gene regulation.
– A microarray is a small glass slide on which cDNAs of many
(or all) genes in an organism have been dotted.
– cDNA is made using mRNAs present under certain conditions
(or in a certain tissue) and labeled with fluorescent dyes.
– Then, the labeled cDNA are hybridized to the microarray and
the fluorescence determined.
• There is a nice animation describing this at:
– http://www.bio.davidson.edu/courses/genomics/chip/chip.html
– Does this examine transcriptional regulation?
Examining Transcriptional Regulation
• This basic method was extended for the Gal4p study
that we have been discussing discussed.
– For this study, the researchers tagged the Gal4p protein so the
could purify from the cell.
– Then, they chemically cross-linked it to DNA and purified it.
– This allowed them to purify the DNA that Gal4p was bound to
in the cell.
– The DNA that Gal4p was bound to in the cell was labeled and
used to probe the microarray.
– Does this examine transcriptional regulation?
Examining Transcriptional Regulation
• This study established several interesting facts:
– The Gal4p binding sites in the DNA are sometimes bound by
Gal4p in the absence of galactose, others are bound only in
the presence of galactose.
– So the trigger is more complex than simply whether or not the
Gal4p protein can bind.
– This more complex regulation involves Gal80p, an inhibitor.
Two possible models
for regulation of the
Gal4p-Gal80p complex
by galactose.
The models differ only
in the exact binding
sites for Gal80p.
How do Eukaryotic Transcriptional
Regulators Work?
• There are a few specific types of proteins that act to
increase transcriptional activity:
– Many proteins have an acidic domain.
• Surprisingly, these “acid-blob” proteins often require a
hydrophobic residue embedded in an acidic region.
• Both Gal4p and the herpes simplex virus VP16 protein (an
transcriptional regulator for this virus) have acid blobs.
– Glutamine-rich and Proline-rich transcriptional activation
domains have been characterized.
• These protein regions activate transcription when
fused to other DNA-binding domains.
– Alternatively, they can be recruited by protein-protein
interactions - e.g., a DNA-binding protein binds the enhancer,
and it contains a region that recruits and acid-blob protein.
Using Eukaryotic Transcriptional Regulators
• The yeast 2-hybrid system exploits these features of
eukaryotic transcription factors to examine proteinprotein interactions.
– The DNA-binding and transcription activating regions of Gal4p
can be separated.
– Interestingly, if you fuse one protein to the Gal4p DNA-binding
domain (BD) and a second protein that it interacts (physically)
with to the Gal4p transcriptional activating domain (AD), one
can see transcriptional activation:
How do Eukaryotic Transcriptional
Regulators Work?
• Another interesting phenomenon that is sometimes
seen with transcription factor is SQUELCHING.
– Overexpression of transcription activators like Gal4p can
result in a general inhibition of transcriptional activity.
– How does this happen?
– Presumably, specific transcription factors like Gal4p act by
recruiting “basal” transcription factors.
• In fact, some basal factors that physically interact with these
transcription activating domains have been found.
• Basal factors are factors involved in recruiting RNA polymerase II
to a large number of promoters.
– So overexpressing proteins with these transcription activating
domains can actually turn gene expression off, by competing
for these factors.
How do Eukaryotic Transcriptional
Regulators Work?
• At least one way is by altering the packing of DNA into
chromatin.
• The role of chromatin structure in the regulation of
transcription is an area of very active investigation.
• However, two important factors that play clear roles in
transcriptional regulation are known:
– DNA METHYLATION - A subset of cytosine (C) residues are
modified by methylation.
– HISTONE ACETYLATION - Histones can be modified by
acetylation.
Chromatin
• Remember, DNA in
eukaryotes packs into
CHROMATIN.
• HISTONES form the
NUCLEOSOME, which
DNA loops around.
• EUCHROMATIN - less
compact; actively
transcribed
• HETEROCHROMATIN more compact;
transcriptionally
inactive.
– Heterochromatin can be
either constitutive or
facultative.
DNA Methylation
• Genes that are transcriptionally inactive are often
METHYLATED.
– In eukaryotes, cytosine residues are modified by methylation.
NH 2
NH 2
CYTOSINE
CH 3
N
O
N
N
H
O
N
H
METHYL-C
• Typically, the sites of methylation are CG dinucleotides
(vertebrates).
– This allows maintenance through replication.
Histone Acetylation
• HISTONES in transcriptionally active genes are often
ACETYLATED.
• Acetylation is the modification of lysine residues in
histones.
– Reduces positive charge, weakens the interaction with DNA.
– Makes DNA more accessible to RNA polymerase II
• Enzymes that ACETYLATE HISTONES are recruited to
actively transcribed genes.
• Enzymes that remove acetyl groups from histones are
recruited to methylated DNA.
– There are additional types of histone modification as well,
such as methylation of the histones.
Genetic Imprinting
• Remember that DNA methylation can be maintained
through replication.
• This allows the packing of chromatin to be passed on just like a gene sequence.
– However, differences in chromatin packing are not as stable as
gene sequences.
• Heritable but potentially reversible changes in gene
expression are called EPIGENETIC phenomena
– Vertebrates use these differences in chromatin packing to
IMPRINT certain patterns of gene regulation.
– Some genes show MATERNAL IMPRINTING while other show
PATERNAL IMPRINTING.
• The alleles of some genes that are inherited from the
relevant parent are methylated, and therefore are not
expressed.
Prader-Willi &
Angelman Syndromes
• Both of these genetic disorders are caused by
deletion of a region of chromosome 15.
• However, the syndromes differ:
– Prader-Willi Syndrome - obesity, mental retardation,
short stature. (abbreviated PWS)
– Angelman Syndrome - uncontrollable laughter, jerky
movements, and other motor and mental symptoms.
(abbreviated AS)
• Syndrome that develops depends upon the
parent that provided the mutant chromosome.
PWS
AS
PWS
Mouse
model
AS
Mouse
model
From Annu Rev Genomics & Hum Genet
Prader-Willi & Angelman Syndromes
• Prader-Willi Syndrome - develops when the
abnormal copy of chromosome 15 is inherited
from the father.
• Angelman Syndrome - develops when the
abnormal copy of chromosome 15 is inherited
from the mother.
• The differences reflect the fact that some loci
are IMPRINTED - so only the allele inherited
from one parent is expressed.
– The region contains both maternally and paternally
imprinted genes.
Methylation and Gene Regulation
• For imprinted genes, the pattern of gene
regulation is dependent upon the parent
that donated the chromosome.
– The methylation pattern is “reprogrammed”
in the germ line.
• There are other examples of methylation
changes the regulate gene expression.
– In mammals, one of the two X chromosomes
in females is inactivated.
– The inactivated X is methylated.