Download CHAPTER 31

Page 1 of 16
Chapter 31 Notes
Biochemistry 461
Fall 2010
CHAPTER 31: CONTROL OF GENE EXPRESSION
LECTURE TOPICS
!
Differential Gene Expression in Procaryotes and Eucaryotes
!
Regulation of Gene Expession
!
Transcriptional Regulation by DNA-Binding Proteins (E.coli Lactose
Operon)
!
Transcriptional and Postranscriptional Regulation (trp Operon
Attenuation; ferritin/transferrin receptor in eucaryotes)
!
Helix-turn-helix motif of Procaryotic DNA Binding Proteins
!
Eucaryotic Gene Regulation
!
Complexity of Genomes
!
Elaborate Mechanisms of Regulation
!
Transcription Activation/Repression mediated by Protein-Protein
Interactions
OVERVIEW: PROCARYOTIC GENE EXPRESSION
!
Once again - Bacteria (E. coli) are most well studied with regard to regulation of
gene expression. Differences in rates of synthesis of some proteins varies over
a 1000-fold range in response to changes in quantity and quality of metabolites,
nutrients, environmental challenges, etc.
!
Regulation of bacterial gene expression occurs most often at the level of
transcription. Genes are often clustered in operons, all of whose genes are
transcribed coordinately in a single mRNA molecule. These genes can be
expressed at all times in the cell (constitutive genes) or they can be under the
control of repressor and/or activator proteins (inducible genes).
Page 2 of 16
!
Many regulatory proteins which bind to DNA have a helix-turn-helix-motif in
common.
!
Examples of operons whose transcriptional regulation is well known are the
lactose and tryptophan operons of E. coli.
!
Gene expression is also regulated at the translational level. [Exs.: trp operon in
E. coli; ferritin and transferrin receptor in eucaryotes.
OVERVIEW: EUCARYOTIC CHROMOSOMES AND GENE EXPRESSION
!
Eucaryotic chromosomes are larger, have higher degrees of structural order,
and a more complex composition than procaryotic chromosomes. The human
genome (4x109 bp = 1 meter) is 1,000 times the size of that of E. coli. DNA
(4x106 bp = 1.4 mm). Replication and gene expression is more complex than the
procaryotic model.
!
Eu
caryotic chromosomal DNA is wound around histones in complexes called
nucleosomes. The entire chromosome also contains many other proteins in a complex
matrix called chromatin.
!
A small fraction (1- 2%) of eucaryotic DNA is genes which code for
proteins. There is an abundance of repetitive, non-coding sequences which
comprise a significant fraction of eucarotic genomes.
!
Eucaryotic gene expression is regulated primarily at the level of
transcription. Transcriptionally active regions of chromosomes are extrasensitive to DNase digestion and have reduced levels of cytosines which have
been methylated. Expression of genes in these chromosomal regions is
regulated by transcriptional factors.
Page 3 of 16
!
Translational regulation occurs in iron metabolism genes.
!
Some developmentally-controlled genes in insects and mammals possess a
sequence called the homeo box, which codes for a 60 amino acid long DNAbinding polypeptide domain (homeo domain) which may be involved in
regulating expression of these genes.
DIFFERENTIAL GENE EXPRESSION IN PROCARYOTES AND EUCARYOTES
!
Some procaryotic genes are differentially expressed in response to different
environmental variables (temperature, nutrients, etc)
!
Many genes are expressed at different times and in different locations (tissues,
organs) in a eucaryotic organism during it’s lifespan. (Table 31.1)
Page 4 of 16
REGULATION OF PROCARYOTIC GENE EXPESSION
TRANSCRIPTIONAL REGULATION BY DNA-BINDING PROTEINS: E.coli Lactose
Operon
!
To use lactose as a sole carbon source, E. coli synthesizes $-galactosidase
(thousands/cell) only when cells are grown on lactose. The enzyme is inducible
by conversion in the cell of lactose to allolactose (a product of a reaction
catalyzed by the 10 or so $-galactosidase molecules present in the cell prior to
induction). (p.868 and Fig.31.1)
$ - galactosidase induction:
(Lactose or IPTG in lab)
To measure $-galactosidase activity in the lab: X-gal cleavage gives galactose and a
blue colored product that cna be quantitated. (Fig.31.1)
Page 5 of 16
!
The $-galactosidase gene is in an operon which contains three structural
genes (z, y, a), two control sites called the promoter (p) and operator (o), and a
regulatory gene (i). Since there is more than one structural gene (cistron), the
lactose operon is called a polycistronic operon. (Fig.31.3)
!
The regulatory gene produces a repressor protein that normally (no lactose in
medium) binds to the operator, preventing RNA polymerase from transcribing
genes z, y, and a. An inducer(allolactose or IPTG), forms a repressor-inducer
complex that cannot bind to the operator, permitting transcription. (Fig.31.8)
Page 6 of 16
!
The lac repressor binds to an inverted
repeat (operator) that is nearly a
palindromic sequence with dyad
symmetry. [Fig. 36-9]
!
lac repressor DNA interactions
(Figs.31.5, 6)
!
An activator (CAP) protein binds cyclic AMP (levels increase when glucose in
medium is low) and this cAMP-CAP complex bind upstream of the promoter site
and stimulates transcription (50X more than without CAP). This overcomes the
lac's lack of a strong promoter consensus sequence. (Fig.31.10) [Fig. 36-13]
!
CAP-RNA polymerase binding sites are adjacent. RNA polymerase-repressor
binding sites overlap. Repressor sterically interferes with RNA polymerase
binding, while CAP facilitates binding of RNA polymerase. A CAP dimer (with
cAMP) binds to DNA in the major groove and bends the DNA by 94 degrees!!!
(Fig.31.11)
Page 7 of 16
HELIX-TURN-HELIX MOTIF OF GENE REGULATING PROCARYOTIC DNA BINDING
PROTEINS
!
Three dimensional models of cro, lambda repressor, and CAP show that these
polypeptides share an "-helix-$-turn-"-helix motif which is involved in
specific protein-DNA interactions. These proteins occur as dimers which
interact with specific DNA sequences via binding of an "-helical polypeptide
domain with the major groove of a symmetrically-oriented recognition site
spanning one turn of a B-DNA helix. [Fig. 36-1,27,29]
!
$-strand DNA interactions are basis for
recognition in methionine repressor
(Fig.31.13) (recall also eucaryotic TATAbox binding protein)
Page 8 of 16
PROCARYOTIC TRANSCRIPTIONAL AND POSTRANSCRIPTIONAL REGULATION
TRYPTOPHAN OPERON REGULATION: ATTENUATION
!
Transcription and translation interact to regulate the tryptophan operon.
!
The tryptophan operon has several structural genes for proteins involved in
tryptophan synthesis. There is an operator site where tryptophan repressor,
complexed with tryptophan (a corepressor), binds, inhibiting transcription.
[Figs. 36-31,32]
!
A leader (L) sequence is 5' to an attenuator sequence [Fig. 36-34]. The leader
codes for a polypeptide [Fig. 36-35] whose synthesis occurs at high cellular
tryptophan levels and whose synthesis is inhibited at low tryptophan levels. The
leader polypeptide has two tryptophan codons. (Fig.31.34)
!
When cellular tryptophan concentration is high, leader translation is enhanced
and transcription is inhibited by the attenuator's secondary structure, which looks
like that of a terminator
(i.e., a GC-rich region with
two-fold symmetry
followed by U's in the
mRNA which makes a
stem-loop structure).
(Fig.31.34) [Fig. 36-36]
!
When tryptophan levels
are low, leader translation
is slow, the tryp mRNA
does not assume a
terminator-like
appearance, and the
tryptophan operon is
transcribed (Fig.31.35)
[Fig. 36-36].
Trp Operon Attenuation Mechanism
High Trp (W)
fast translation
3-4 stem/loop stops transcription
Low Trp (W)
slow translation
2-3 stem/loop allows
transcription
Page 9 of 16
TRANSLATIONAL REGULATION IN EUCARYOTES: Proteins synthesis from
ferritin and transferrin receptor mRNAs (Figs.31.38,39)
!
IRE (iron response element) binding protein blocks translation of ferritin
mRNA.
!
Transferrin receptor mRNA has several IREs at 3'-end. IRE-binding protein
located on these IRE’s stabilizes mRNA and does not inhibit translation.
Page 10 of 16
EUCARYOTIC GENE REGULATION
COMPLEXITY OF GENOMES: EUCARYOTIC CHROMOSOMES
!
SIZE: Large genomes (1 meter long in human
genome) linear molecules. (Figs.31.14.15) [Table
37-1]
!
COMPOSITION: Contain five types of basic
proteins called histones which have lots (25%) of Arg and Lys residues and are
11 to 21 Kd in mass. The histones are frequently modified by acetylation,
methylation, Phosphorylation, etc. These modifications may relate to DNA
packaging or availability for replication or transcription [Table 37-2]. Histone
amino acid sequences and structures are highly conserved (especially
histones H3 and H4) in all eucaryotes, suggesting that the role of histones
was established early during eucaryotic evolution. (H2, 3, 4 structures,
Fig.31.17)
STRUCTURE:
Page 11 of 16
!
Nucleosomes [Fig. 37-5, 8] are repeating units of chromatin which consist of
core particles (a histone octamer around which is wrapped 140 base pairs of
DNA) connected by linker DNA (20 to 50 base pairs). The core particles contain
two molecules each of histones H2A, H2B, H3 and H4 and one molecule of
histone H1 binds to the outside of the core particle. The DNA wound around the
core particle forms a 1-3/4 turn left-handed superhelix [Fig.31.16, Fig. 33-8].
Nucleosomes condense DNA into a smaller space (packing ratio 7:1). Further
DNA packing must occur in cells, since metaphase chromosomes have a
packing ratio of 1000:1. (Fig.31.18)
Page 12 of 16
!
A protein scaffold of non-histone proteins
provides a higher order structure and higher
packing ratio than that of just nucleosomes.
These proteins include topoisomerase II which
suggests that changes in supercoiling are
important in dynamic changes of in chromosomal
DNA structure and function during the cell cycle.
[Fig. 37-11, and Lehninger, Fig. .23-20]
Page 13 of 16
REPETITIVE VS. SINGLE COPY DNA
!
Renaturation analysis revealed that eucaryotic chromosomal DNA has a large
fraction of several types sequences (satellites, Alu sequences) that are repeated
up to a million times. Results also showed that there was only a small fraction of
unique protein-coding sequences. [Table 37-6]
!
Ribosomal RNA genes are tandemly repeated several hundred times and can
be amplified to 2X106 copies during development of Xenopus oocytes to up to
75% of total cellular DNA. [Amplification provides enough genes to rapidly
transcribe rRNA genes to get 1012 ribosomes/oocyte.]
!
Histone genes are clustered, repeated, lack introns, and have mRNA which is
not polyadenylated.
!
Many major (and minor) cell proteins are coded by single copy genes. For
major cellular proteins, transcription rates, mRNA half-life, and use of mRNA for
multiple rounds of translation can give up to 109 protein molecules from just one
gene copy (silk fibroin).
!
the human genome is 1000X the size of the E. coli chromosome, but only 1-2%
codes for proteins. Thus, the actual unique human genome size is only
about 50X that of E. coli.
Page 14 of 16
REGULATION OF EUCARYOTIC GENE EXPRESSION: Elaborate Mechanisms
!
Regions of chromosomes which are transcriptionally active are less
condensed (form puffs in Drosophila [Fig. 37-29] are undermethylated, and
are hypersensitive to DNase I (mostly at the 5'-ends of genes). These
characteristics are tissue-specific and developmentally regulated.
!
Methylation of cytosines [Fig. 37-30]
in chromosomal DNA is associated
with low transcriptional activity, in
general. Azacytosine prevents
methylation, so transcrption is more
active.
!
Not all possible regulatory sites on
chromosomes in a given cell/tissue
type may be activated at the same
time. For instance, yeast GAL4
protein activates genes for proteins
required in galactose utilization as a
carbon source. But, of 4000 possible
specific binding sequences, only 10
actually have GAL4 bound and only 10
genes must be activated for galactose
utilization.
Page 15 of 16
!
Disruption of chromatin structure occurs when transcription activator
proteins bind to enhancer sequences. Many enhancer sequence elements are
known. The glucocorticoid enhancer is one example (Ch.28 Notes, p.13). The
muscle creatine kinase gene is another in which 3 copies of one enhancer
sequence occurs and two other enhancer sequences exist. At least 3 different
regulatory proteins are required to bind to all three of these different enhancers.
Often, binding of these proteins disturbs the chromatin structure to expose the
gene DNA to allow transcription. (Figs.31.20,21)
TRANSCRIPTION ACTIVATION/REPRESSION MEDIATED BY PROTEIN-PROTEIN
INTERACTIONS
!
TRANSCRIPTIONAL ACTIVATORS: We already know about transcription
factors and enhancer sequences (see Chapter 28). Transcriptional activators
bind to specific activator sequences (enhancers) and these proteins have in
common at least two conserved
functional domains - one which
binds to DNA and one which
activates transcription (Fig.31.32;
Fig.37-31] In addition, other
conserved domains may be present,
depending on the specific activator.
Page 16 of 16
!
The family of nuclear hormone binding receptors (active as dimers of identical
subunits) has an additional ligand-(hormone) binding domain. The DNAbinding domains of nuclear hormone receptor proteins possess globular
structural domains in which four cysteines are tetrahedrally coordinated with a
divalent zinc ion. Two of these zinc clusters are present on each subunit and
they stabilize the structure of of the dimer. Each subunit of the dimeric steroid
receptor has an "-helix which recognizes and binds to the major groove of the
DNA sequence of the steroid response element. (Fig.31.22)
Fig. 37-35
Fig.31-22
Fig. 37-36