Prokaryotic Gene Expression Mechanisms • • • • • • • •
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 1 2 3 4 5 6 7 8
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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