Samenvatting
Summary - Genetics (AB_1135) - part 2
- Instelling
- Vrije Universiteit Amsterdam (VU)
Summary of chapter 2, 4, 5, 6, 7, 8, 14, 19, 24, 25 of the course genetics (AB_1135).
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Summary Genetics Part 2Lecture 11 Gene Regulation in Bacteria (CH14)
Enzyme adaptation = particular enzyme appears in the cell only after the cell has been
exposed to the enzyme’s substrate
Operon concept = regulatory unit, consisting of few structural genes under control of 1
promoter and terminator
- Encodes polycistronic (poly-ORF) mRNA: contains coding sequence 2 or more
structural proteins → coordinately regulate group of genes that encode proteins
involved in same process / function
- Promoter, operator, terminator, structural genes
1. Catabolism: inducible (lac operon)
- Inducer: substance to be broken down (or a related compound)
2. Anabolism: repressible (trp operon)
- Inhibitor or corepressor: product of the operon (small molecule)
Allolactose: inducer, consisting of D-galactose and D-glucose
Lac operon
Promoter region, DNA elements
- Promoter: bound by RNA polymerase
- Operator: bound by lac repressor protein
- CAP site: bound by Catabolite Activator Protein (CAP)
Structural genes
- LacZ: encodes Beta-galactosidase
+ Enzymatically cleaves lactose and lactose analogues
+ Converts lactose into allolactose (isomer)
- LacY: encodes lactose permease
+ Membrane protein required for transport of lactose and analogues
- LacA: encodes transacetylase
+ Covalently modifies lactose and analogues
+ Suggested to help detoxification caused by excess lactose/analogues by
preventing their cellular re-entry
LacI gene: encodes regulatory protein = lac repressor
- Not part of lac operon
, - Own promoter, i promoter
- The lac repressor protein functions as a homo-tetramer
- Only small amount of protein needed to repress lac operon (10 repressors per cell)
Lac Operon regulation
1. Inducible, negative control mechanism by lac repressor
- No lactose: repressor active → binds operator → transcription inhibited
- Lactose present: some gets converted in allolactose (by B-galactosidase) →
binding repressor → conformational change → transcription
2. Catabolite repression by lac activator protein (CAP, catabolite activator protein)
- Glucose levels low, lactose present: stimulate enzyme adenylyl cyclase →
produce cAMP → cAMP binds CAP → CAP binds promoter region lacP of lac
operon → activate RNA polymerase → transcription enzymes degradation
lactose
- Presence glucose and lactose: adenylyl cyclase activity is low → cAMP is low
in cell → CAP not bound by cAMP → low affinity CAP binding site without
cAMP → no CAP present at CAP binding site → RNA pol not recruited → no
transcription
Cis- and Trans-effects: interaction between regulatory proteins and DNA sequences
1. Cis-effect: DNA sequence that exerts effect on gene to which physically linked
- Example: The lac operator, lac promoter
2. Trans-effect: regulatory proteins that bind cis-acting elements
- Example: The action of the lac I repressor on the lac operon
Merozygote: partial diploid (by transformation) → 2 copies lac operon, one can be mutated
- Complementation: trans-acting, functional protein encoded by second normal allele
can take over the function of the defective allele
Mutant repressor proteins
- i^S = super repressed → can not bind inducer and therefore remain bound to
operator → constitutive repressor
, - i^Q = quantity → produces much more repressor than normal I gene (promoter
up-mutation) → may need more of inducer allolactose to switch on lac operon
Diauxic growth, 2 growth phases: sequential use of 2 sugars by bacteria
- When exposed to glucose and lactose → glucose used first → catabolite repression
prevents use of lactose
- Glucose depleted → catabolite repression is alleviated and lac operon is expressed
Lac Operons
- If all three are present: Max. repression
- If 2 are present, intermediate level of repression
>> Identification and function of multiple lac operon operators
Tryptophan Operon: involved in biosynthesis of amino acid tryptophan
- Enzymes biosynthesis: Genes trpE, trpD, trpC, trpB and trpA
- Regulation trp operon
+ trpR: Encodes the trp repressor protein (functions in repression)
- Low tryptophan levels: transcription
- High tryptophan levels: repression transcription
+ trpL: Encodes short peptide called Leader peptide (functions in attenuation)
- High tryptophan levels: transcription stops at attenuator sequence →
inhibits further production/synthesis of tryptophan
- Back up repression system: p-independent terminator (uracil rich)
Three possible scenarios:
1. No transcription-translation coupling: most stable form of mRNA (all loops) → no
ribosome → transcription terminated just past trpL gene
2. Low levels of tryptophan: only 2-3 stem loop forms → ribosome present (insufficient
amount of tRNA trp) → slow translation
3. High levels of tryptophan: only 3-4 stem loop forms → ribosome present, sufficient
tRNA trp → fast translation until region 2 (stop codon) → transcription terminates
Translational regulation
Proteins inhibit translation → translational repressors
1. Binding next to Shine-Dalgarno sequence and/or start codon → hinder
ribosome from initiating translation
2. Binding outside Shine-Dalgarno/start codon region → stabilize mRNA
secondary structure that prevents initiation (antisense RNA)
Post-translational regulation
Feedback inhibition: final product in pathway can inhibit enzyme that acts early in pathway
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