Biology 457 & 557 - Week 6
General mechanisms which control transcription
1) primary DNA modification correlated with genetic regulation is methylation
w in mammals, -CH3 groups added to
w gene inactivation by methylation of cytosines places methyl groups into the
w also possible that certain regulatory proteins recognize and bind methylated
DNA
w inactive genes are highly methylated in CG islands
w 1 of 2 X chromosomes in female mammalian somatic (body) cells is almost
completely inactivated
inactive heterochromatin
w methylation patterns of globin genes best studied vertebrate model
2) chemical modifications of histones by acetylation and phosphorylation
w histones organize DNA into nucleosomes
- octamer of H2A, H2B, H3, H4 wrapped with ~165 bp DNA (~2 turns)
- H1 binds in region where DNA enters/exits the nucleosome
(stabilizes DNA)
O
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w acetylation (add -C-CH3) of lysine residues by acetyltransferase
- this is reversible by
w core histones are acetylated in regions that interact with DNA
w nucleosomes unfold and regulatory proteins/transcription factors
w may be complete unfolding/partial unfolding
w recently found that a component of the basal transcription factor TFIID
w phosphorylation of H1 converts chromatin into inactive heterochromatin
- induces packing association
- attraction of H1 molecules
- dephosphorylation
CONCLUSIONS:
ä THESE CONTROL MECHANISMS I HAVE DESCRIBED ACT IN CONCERT TO
TURN GENES ON AND OFF AT THE PROPER TIMES AND IN THE PROPER
PLACES USING THE CORRECT COMBINATION OF REGULATORY
ELEMENTS AND SEQUENCES
ä CONTROLLING GENE ACTIVITY PROPERLY IS IMPORTANT FOR
EMBRYONIC DEVELOPMENT THROUGH MAINTENANCE OF ADULT
ACTIVITIES AND IS A VERY COMPLEX PROCESS
because bacteria normally live in a constantly changing environment, most of
their regulatory controls are easily reversible systems
very few bacterial regulatory pathways lead to permanent cellular changes
bacterial genes are organized in operons…..
each operon is controlled as a unit by one or more promoters
the 5’ flanking regions of bacterial operons are recognized and bound by
regulatory proteins called
repressors adjust the rate of initiation
repressor-regulated operons are characterized by increased transcription
activators work in an opposite fashion and adjust their promoters upward
from a low base level
operons are also regulated by sigma factors
these factors have the same overall effect as activators
many operons are controlled by more than one regulatory mechanism
the result is a complex network of superimposed control
best studied example of this is the lac operon
there are 3 genes in this operon (transcriptional unit) coding for 3 enzymes
involved in lactose metabolism
this lac operon is controlled by a regulatory protein called the lac repressor
this repressor is encoded by a gene separate from the lac operon
in an active form, the repressor binds to a site within the promoter
when the repressor is bound to the operator, RNA polymerase can’t bind
tightly
so the repressor binds when no lactose is present
the lac repressor also has a binding site
this binding induces a conformational change in the repressor
RNA polymerase can now bind and transcribe the operon
this type of repressor system ensures that the enzymes of the pathway are not
made unless they are required
the repressor-based mechanism can also reduce the synthesis of the enzymes of a
pathway when a product of the pathway is present in the medium
ex: trp operon,
this system also has a repressor, trp repressor, which has 2 binding sites, one
for the promoter of the trp operon and the other one for tryptophan
however, unlike the lac operon repressor,
in this form, the repressor has no affinity for the trp promoter
this is the situation when the supply of tryptophan is low
if tryptophan becomes available or if cellular levels of the amino acid are high
because of synthesis by this pathway, then excess tryptophan can bind to the
repressor
thus, when tryptophan is available the pathway is off
a few more details of repressors:
in the case of the lac repressor, its 3-D structure has been determined
the tetramer contains helix-turn-helix motifs
most other repressors are homodimers
which contain alpha helices
the genes coding for repressors are also operons
the operons encoding most repressors are autoregulated
as the quantity of repressor molecules increases
autoregulation ensures that there are always some repressor molecules
mention a few words on activators:
typically, activators control groups of operons
activators are also autoregulated
have found positive regulators of the lac and 2 other operons, the gal and ara
operons
ex: cAMP receptor protein (CRP) or catabolite activator protein (CAP)
CRP (CAP) is made in inactive form
when glucose concentration is low
combination with cAMP induces a conformational change
this active cAMP-CRP (CAP) molecule, in the presence of RNA polymerase, can
then bind to the operators of the lac, gal and ara operons simultaneously
activation of these operons allows the production of enzymes which can
metabolize these other sugars
if glucose then becomes available again, the enzyme converting ATP to cAMP is
inactivated