Transcript Chapter 21 (Part 2)

Chapter 21 (Part 2)
Transcriptional Regulation and
RNA Processing
Gene Expression
• Constitutive – Genes expressed in
all cells (Housekeeping genes)
• Induced – Genes whose expression
is regulated by environmental,
developmental, or metabolic signals.
Regulation of Gene Expression
RNA Processing
5’CAP
Active
enzyme
Post-translational
modification
mRNA
AAAAAA
RNA Degradation
Protein Degradation
Transcriptional Regulation
• Regulation occurring at the initiation of
transcription.
• Involves regulatory sequences present
within the promoter region of a gene
(cis-elements)
• Involves soluble protein factors (transacting factors) that promote (activators)
or inhibit (repressors) binding of the
RNA polymerase to the promoter
Cis-elements
• Typically found in 5’ untranscribed
region of the gene (promoter
region).
• Can be specific sites for binding of
activators or repressors.
• Position and orientation of cis
element relative to transcriptional
start site is usually fixed.
Enhancers
• Enhancers are a class of cis-elements
that can be located either upstream or
downstream of the promoter region
(often a long distance away).
• Enhancers can also be present within the
transcribed region of the gene.
• Enhancers can be inverted and still
function
5’-ATGCATGC-3’ = 5’-CGTACGTA-3’
Activators and Repressors
• Activators and
repressors- Bind to
specific cis-elements
• Promote or inhibit
assembly of
transcriptional
initiation complex.
• Co-activators and corepressors – bind to
proteins associated
with cis-elements.
co-A
A
RNAP
+1
RNAP
co-R
R
+1
Structural Motifs in DNA-Binding
Regulatory Proteins
• Crucial feature must be atomic contacts between
protein residues and bases and sugar-phosphate
backbone of DNA
• Most contacts are in the major groove of DNA
• 80% of regulatory proteins can be assigned to one
of three classes: helix-turn-helix (HTH), zinc finger
(Zn-finger) and leucine zipper (bZIP)
• In addition to DNA-binding domains, these proteins
usually possess other domains that interact with
other proteins
The Helix-Turn-Helix
Motif
• contain two alpha
helices separated by
a loop with a beta
turn
• The C-terminal helix
fits in major groove
of DNA; N-terminal
helix stabilizes by
hydrophobic
interactions with Cterminal helix
The Zn-Finger Motif
Zn fingers form a folded beta strand and an alpha helix that
fits into the DNA major groove.
The Leucine Zipper Motif
• Forms amphipathic
alpha helix and a
coiled-coil dimer
• Leucine zipper proteins
dimerize, either as
homo- or heterodimers
• The basic region is the
DNA-recognition site
• Basic region is often
modeled as a pair of
helices that can wrap
around the major
groove
Transcription Regulation in
Prokaryotes
• Genes for enzymes for pathways are grouped in
clusters on the chromosome - called operons
• This allows coordinated expression
• A regulatory sequence adjacent to such a unit
determines whether it is transcribed - this is
the ‘operator’
• Regulatory proteins work with operators to
control transcription of the genes
Induction and Repression
• Increased synthesis of genes in response
to a metabolite is ‘induction’
• Decreased synthesis in response to a
metabolite is ‘repression’
Binding of some trans-factors is
regulated by allosteric modification
lac operon
• Lac operon – encodes 3 proteins involved in
galactosides uptake and catabolism.
•
lacY = Permease – imports galactosides (lactose)
•
lacZ = b-galactosidase – Cleaves lactose to
glucose and galactose.
•
lacA = thiogalactoside transacetylase – function
unclear
• Expression of lac operon is negatively regulated by
the lacI protein
Lactose (Lac) Operon
• Diauxic growth
• Operon organization
• Negative and positive regulation
– Repressor (lacI)
– CAP (crp)
Glucose is E. coli’s
primary carbon
source.
But.. it can grow
on different carbon
sources.
Diauxic growth of E. coli on a mixture of
lactose + glucose.
• When Glucose runs out but
Galactose is present, a set
of genes (lac operon) are
induced to break down
Lactose
The lac I protein
• The structural genes of the lac operon are
controlled by negative regulation
• lacI gene product is the lac repressor
• When the lacI protein binds to the lac operator
it prevents transcription
• lac repressor – 2 domains - DNA binding on Nterm; C-term. binds inducer, forms tetramer.
Operator and RNA Polymerase Bind at Overlapping Sites
Inhibition of repression of lac
operon by inducer binding to lacI
• Binding of inducer to lacI cause allosteric change that
prevents binding to the operator
• Inducer is allolactose which is formed when excess
lactose is present.
Inducer : Allolactose,
produced by side
reaction of lacZ
Lehninger: Principles of Biochemistry, 3rd Ed.
IPTG is a Gratuitous Inducer
IPTG is a synthetic
molecule
• Synthetic molecule
Not metabolized by lacZ
Used by molecular
biologist to induce
protein expression using
the lac promoter
Binding of inducer
causes allosteric
change that prevents
binding to operator
LacI is always
bound to
operator unless
the inducer is
present
Although some
leaky expression
does occur
Keeps lac operon
in pressed state
until it is
needed
Repression of
the Tryptophan
operon:
A variation of
the theme
Catabolite Repression of lac Operon
(Positive regulation)
• When excess glucose is present, the lac operon is
repressed even in the presence of lactose.
• In the absence of glucose, the lac operon is induced.
• Absence of glucose results in the increase synthesis of
cAMP
• cAMP binds to cAMP regulatory protein (CRP) (AKA CAP).
• When activated by cAMP, CRP binds to lac promoter and
stimulates transcription.
Molecular Cell Biology, 4th Edition, Lodish et. al. (2000)
Why does the Lac Operon
need an activator?
Lac promoter has lousy promoter!!!
Post-transcriptional
Modification of RNA
• tRNA Processing
• rRNA Processing
• Eukaryotic mRNA Processing
tRNA Processing
•tRNA is first transcribed by RNA
•Polymerase III, is then processed
•tRNAs are further processed in the chemical
modification of bases
rRNA Processing
•Multiple rRNAs are originally transcribed as single
transcript.
•In eukaryotes involves RNA polymerase I
•5 endonuclases involved in the processing
Processing of Eukaryotic mRNA
5’ Capping
• Primary transcripts (aka pre-mRNAs or
heterogeneous nuclear RNA) are usually first
"capped" by a guanylyl group
• The reaction is catalyzed by guanylyl
transferase
• Capping G residue is methylated at 7position
• Additional methylations occur at 2'-O
positions of next two residues and at 6amino of the first adenine
• Modification required to increase mRNA
stability
3'-Polyadenylylation
• Termination of transcription occurs only
after RNA polymerase has transcribed
past a consensus AAUAAA sequence the poly(A)+ addition site
• 10-30 nucleotides past this site, a
string of 100 to 200 adenine residues
are added to the mRNA transcript the poly(A)+ tail
• poly(A) polymerase adds these A
residues
• poly(A) tail may govern stability of the
mRNA
Splicing of Pre-mRNA
• Pre-mRNA must be capped and polyadenylated before
splicing
• In "splicing", the introns are excised and the exons are
sewn together to form mature mRNA
• Splicing occurs only in the nucleus
• The 5'-end of an intron in higher eukaryotes is always
GU and the 3'-end is always AG
• All introns have a "branch site" 18 to 40 nucleotides
upstream from 3'-splice site
Splicing of Pre-mRNA
• Lariat structure forms
by interaction with
5’splice site G and 2’OH
of A in the branch
site.
• Exons are then joined
and lariot is excised.
• Splicing complex
includes snRNAs that
are involved in
identification of splice
junctions.