Lesson 2.4 - trp operon & Lenski experiment Flashcards
Function of 5’ UTR
Regulatory functions; occurs upstream and precedes 1st gene
DNA - Protein Interactions
- Necessary for super-coiling, replication, transcription, & repair
- Twisting creates (2) grooves that allow proteins to contact bases
- Surfaces of A-T & G-C are distinct (physically & chemically)
What is the trp operon?
- Codes for biosynthetic enzymes for L-tryptophan
- 5 genes
- trp A, B, C, D, E
Tryptophan (trp) operon is ________
- repressible
- Degault state is ON (negative feedback)
Biological pathway of Tryptophan biosynthesis
Chorismate to trp A, B, C, D, E to L-tryptophan
(2) levels of regulation for trp operon
- Repressor / co-repressor model
- Attenuation
Repressor / co-repressor model
- Operon is OFF when repressor (TrpR) is bound
- Only binds to operator in high presence of co-repressor (L-tryptophan)
What happens when [Trp] is low? (Repressor / co-repressor model)
- RNA poly. performs transcription of 5 genes in operon
- Polycistronic mRNA translated
What happens when [Trp] is high? (Repressor / co-repressor model)
- Repressor bings to operator, prevents RNA poly.
- 3 active repressors bind to operator
What is an attenuator?
Device with arrangement of resistors that reduce strength of a signal
trp attentuation is influenced by ________.
-
intracellular [Trp]
- Gradually reduces expression of operon as it increases
- Requires transcription & translation coordination
- Form of homeostasis
16S rRNA is found in the….
small ribosomal subunit
Attenuation relies upon formation of _____ in the _______
- hairpin structures; 5’ UTR / leader sequence
- Contains 14 codon sequence w/ 2 codons for tryptophan
- Upstream to trp E gene
- Contains 14 codon sequence w/ 2 codons for tryptophan
What happens when Trp-tRNA is low? (Attentuation)
- Stall at the Trp codons
- Stalled ribosome spans region 1; 1-2 hairpin doesn’t form
- 2-3 hairpin forms & prevents 3-4 hairpin (Attenuator & Terminator)
- Stalled ribosome spans region 1; 1-2 hairpin doesn’t form
What happens when Trp-tRNA is high? (Attenuation)
- No stall at Trp codons
- Leader is fully translated; pauses at STOP codon; blocks region 2
- 3-4 hairpin forms and removes RNA poly.
Most stable secondary structure for trp leader mRNA is when…
… regions 1-2 and 3-4 base-pairs and forms hairpin secondary structures
Attenuation is only possible because…
transcription and translation are coupled
What was the goal of the Lenski experiment?
- To identify mutations that increased fitness (changes in phenotype in long-term evolution)
- Faster gowth rates, increased cell size, increase in DNA repair defects
What was used in Lenski experiment? What was transferred?
- (1988) E. coli strains
- Medium with 139 µM glucose & 1700 µM citrate (Fe acquisition)
- 1 mL of culture transferred to 99 mL of fresh medium (1/100)
What were the results of the Lenski experiment?
-
E. coli could catabolize citrate after 33,000 generations; lead to increased density
- Cells evolved way to produce CitT in aerobic conditions
Function of CitT transport protein
- Antiporter; transport citrate molecules into cell in exchange for succinate molecules
- Passive transport (no ATP); citrate enters Kreb’s cycle
- 1 ATP & 2 NADH (export succinate in return for more citrate)
Negative control of cit operon (before long-term experiment)
- O2 present: repressor protein binds to cit promoter & blocks transcription
- No transcription
cit operon after duplication event
- cit promoter still inhibited by repressor protein
- Genes and promoters in region rearranged
- repositioned rnk promoter (nucleoside diphosphate kinase regulator) facilitates downstream duplicated copy
What can the respositioned rnk promoter do? (after duplication event)
- Transcribe downstream DNA, duplicated copy of citT gene
- O2 present: transmembrane protein made = advantage for transporting energy rich molecules
