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Prokaryotic Gene Expression
Mechanisms
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RNA
Transcription
Prokaryotes vs. Eukaryotes
Mechanisms of Prokaryotic Gene Expression
The Operon Theory
lac constitutive mutants – lac repressor
Operator constitutive mutants – cis vs. trans
Protein binding to the regulatory region
Types of RNA
• mRNA - messenger RNA. Intermediate of
Central Dogma. Encodes protein.
• rRNA - ribosomal RNA. Structural
component of ribosomes. Includes several
molecules of RNA. Serves as “scaffold.”
• tRNA - transfer RNA. Provides translation
from nucleic acid code to amino acid code.
RNA
• RNA has 2’OH - creates more reactive
cis-diol, RNA is more reactive.
• Uracil replaces thymine.
• RNA is normally found single-stranded, but
with internal base-pairing.
Other types
• snRNA - small nuclear RNA.
• scRNA - small cytoplasmic RNA.
• hnRNA - heterogeneous nuclear RNA
(precursor to mRNA).
• Others.
Central Dogma
DNA ----> RNA ----> Protein
This describes the flow of information from DNA into RNA
(most commonly mRNA) through transcription (copying the
same code from one molecule to another), and then expressing the
code into a functional molecule by translation (converting from a
nucleic acid code into an amino acid code).
Enhanced by
Higher Temps
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RNA Polymerase
• Similar in enzymatic function to DNA
polymerases, but-• DOES NOT REQUIRE PRIMER!
• Instead, requires promoter sequence on
DNA.
• Recognition of promoter usually requires
accessory proteins or subunits.
Prokaryotes vs. Eukaryotes
• Eu = “true” karyon = “nucleus” (literally,
“colored body”)
• Eukaryotic cells are compartmentalized
• Prokaryotic cells are not. Therefore,
transcription and translation occur in the
same compartment (and often on the same
RNA molecule)
RNA Polymerase
• Choice of template strand specifies
direction.
• Choice of template strand is specified in
promoter sequence.
• Product RNA is:
– unstable (temporal changes in expression)
– not proofread (discarded)
– malleable (may be altered enzymatically)
Coupled
Transcription-Translation
• Impossible in eukaryotes
• Allows ribosomes to bind RNA as it is
being synthesized.
• This binding may affect transcription.
Polycistronic RNA
• One mRNA may contain several proteincoding sequences.
• Each protein-coding sequence is called a
cistron.
• Resulting RNA is therefore polycistronic.
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E. coli RNA Polymerase
E. coli RNA polymerase
• DNA-dependent RNA polymerase
• Core polymerase contains four subunits,
2 α, 1 β and 1 β’ (α2ββ’)
• MW 380,000
• Core polymerase capable of binding DNA,
transcribing RNA, but lacks specificity.
Sigma (σ) factor
• Transient subunit of E. coli RNA pol.
• Increases affinity* for promoter sequences
106-fold.
• Binds RNA pol, complex binds promoter,
transcription begins, then σ dissociates.
*See Handout on Affinity on Web Site.
Molecular recognition
Sigma factor specificity
• At least 5 σ factors in E. coli
• Most commonly used is 70,000 MW, so is
named σ70
• Another is σ32, which responds to heatshock.
• Each type of σ factor is specific for a class
of genes.
Keq is a measure of strength of
interaction between two molecules
[AB]
=
Keq
[A][B]
See Handout on Web Site for Details!
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DNA-protein signaling
Promoter
• A specific sequence, defined by homology,
point mutations and/or protein binding
specifies a functional location on DNA.
• A protein recognizes this sequence, as
defined by specific binding.
• Specificity is defined by affinity.
• A promoter is the sequence required for
initiation of transcription.
• In prokaryotes, the promoter is also the
specific binding site for RNA pol.
• Promoters were first identified by sequence
homology.
Promoter Sequence Homology
Pribnow Box
5'-TATAAT-3'
• “Consensus” sequence - not found in every
occurrence.
• Strand-specific.
• Usually about -10 bp from start site.
-35 Sequence
Promoter consensus
5'-TTGACAT-3'
• Found about -35 bp.
• Together with Pribnow box, sufficient for
binding RNA pol containing σ70
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Promoter binding of proteins
DNA
“Footprint”
Bound protein
“protects” DNA
from digestion,
leaving “footprint”
on gel.
Definition of promoter by protein
binding (footprinting)
The Operon Theory
• Jacob & Monod - worked in late 50’s, early
60’s. Shared Nobel in 1965.
• Operon - a group of genes regulated as a
group (coordinately regulated)
• Grew out of the study of polycistronic genes
of E. coli.
The lac Operon
Newly-synthesized lac genes
• Lac operon contains three genes:
• E. coli metabolizes several sugars.
• “Prefers” glucose
• When glucose is exhausted,
begins to metabolize lactose
• Requires time.
– lac Z - β-galactosidase
– lac Y - lactose permease
– lac A - thiogalactoside transacetylase
• All three share a single promoter
• All three synthesized from a single mRNA
(polycistronic)
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Gene Regulation Requires
Specific Sequences and Proteins
• Lac gene products normally repressed.
• When glucose exhausted, and in the
presence of lactose, lac genes are induced.
• What are the cis-acting and transacting regulatory elements?
Hypothesis - lacIc
• Since lacIc is a mutation, it must represent a
defective gene product—a protein.
• Jacob & Monod hypothesized that the
normal function of the lacI gene is to
encode a repressor protein.
• lacIc must be a mutant that fails to express
repressor and fails to repress lacZ.
Theory of Gene Regulation
• A specific sequence, defined by homology,
point mutations and/or protein binding
specifies a functional location on DNA.
• A protein recognizes this sequence, as
defined by specific binding.
• Specificity is determined by affinity.
lac constitutive mutants
• Wt E. coli grows
grows blue on
X-gal plates if no
glucose present.
• E. coli mutations
grow blue on
X-gal plates even if
glucose is present
• These mutations
named lacIc
Trans-acting factor
• The lacIc mutation mapped far away from
the lac operon on E. coli genome.
• The wild-type lacI gene is hypothesized to
produce a protein, which may diffuse to
another site on the chromosome and bind a
regulatory sequence.
Regulated gene expression
requires:
• A regulatory DNA sequence; and
• A "machine" (protein) capable of
recognizing the DNA sequence and
passing the signal to, for example, RNA
polymerase. (This protein may have its
binding activity regulated.)
• The protein may be regulated.
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E. coli merodiploid
Lac operon
IPTG binds Repressor
Lac operon
IPTG
+
IPTG
E. coli
Chromosome
Plasmid
When IPTG binds Repressor protein, it
removes Repressor from DNA.
Haploid
Merodiploid
Results of crosses
Number
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2
3
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5
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Genotype
I+O+Z+
I+OcZ+
I+OcZ+/I+O+Z+
I+OcZI+OcZ-/I+O+Z+
I-O+Z+
I-O+Z+/I+O+Z+
I-O+Z+/I+O+Z-
Interpretation
β-galactosidase activity
(lac Z)
-IPTG
+IPTG
+
+
+
-
+
+
+
+
+
+
+
Trans activity of repressor
#7
#3
Repressor binds to
the lacO site, which
overlaps and
interferes with
lacP.
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Repressor binds lacO site
Regulated Affinity
The addition of inducer IPTG decreases the KA of
repressor for lacO to 2 x 1010, but the affinity for random
DNA sequences remains the same. So the specificity of
repressor for lacO drops 3 orders of magnitude (or
1000-fold). Under these conditions, you can calculate that
less than 3% of the lacO sites should have repressor
bound to them (when IPTG is present).
∴ IPTG regulates lac expression by
regulating the affinity of repressor for lacO
Repressor regulated by lactose
Lessons learned from lac operon
• Genes include structural (protein-coding)
sequences and regulatory sequences
(Actually, lac repressor
does not block pol binding,
instead prevents transition
from initiation to
elongation.)
• A basal promoter is usually necessary for gene
expression
• A promoter binds RNA polymerase, when
sequence-specific FACTORs (such as σ subunit)
are included
• Regulatory FACTORs interact with specific
DNA sequences to regulate gene expression
More lessons...
• A FACTOR must bind specific DNA sequences
with higher affinity than random sequences, and
this preferential binding may be influenced by
chemical signals (IPTG or cAMP).
• A FACTOR must somehow signal gene
expression.
• A factor may be gene-specific (lac) or specific for
a group of genes (CAP—see pp. 184-191).
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