Lesson 2.4 - trp operon & Lenski experiment

Question 1

Function of 5’ UTR

Answer 1

Regulatory functions; occurs upstream and precedes 1st gene

Question 2

DNA - Protein Interactions

Answer 2

• 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)

Question 3

What is the trp operon?

Answer 3

• Codes for biosynthetic enzymes for L-tryptophan
• 5 genes
  
  • trp A, B, C, D, E

Question 4

Tryptophan (trp) operon is ________

Answer 4

• repressible
  
  • Degault state is ON (negative feedback)

Question 5

Biological pathway of Tryptophan biosynthesis

Answer 5

Chorismate to trp A, B, C, D, E to L-tryptophan

Question 6

(2) levels of regulation for trp operon

Answer 6

• Repressor / co-repressor model
• Attenuation

Question 7

Repressor / co-repressor model

Answer 7

• Operon is OFF when repressor (TrpR) is bound
  
  • Only binds to operator in high presence of co-repressor (L-tryptophan)

Question 8

What happens when [Trp] is low? (Repressor / co-repressor model)

Answer 8

• RNA poly. performs transcription of 5 genes in operon
• Polycistronic mRNA translated

Question 9

What happens when [Trp] is high? (Repressor / co-repressor model)

Answer 9

• Repressor bings to operator, prevents RNA poly.
  
  • 3 active repressors bind to operator

Question 10

What is an attenuator?

Answer 10

Device with arrangement of resistors that reduce strength of a signal

Question 11

trp attentuation is influenced by ________.

Answer 11

• intracellular [Trp]
  
  • ​Gradually reduces expression of operon as it increases
• Requires transcription & translation coordination
  
  • Form of homeostasis

Question 12

16S rRNA is found in the….

Answer 12

small ribosomal subunit

Question 13

Attenuation relies upon formation of _____ in the _______

Answer 13

• hairpin structures; 5’ UTR / leader sequence
  
  • Contains 14 codon sequence w/ 2 codons for tryptophan
    
    • Upstream to trp E gene

Question 14

What happens when Trp-tRNA is low? (Attentuation)

Answer 14

• 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)

Question 15

What happens when Trp-tRNA is high? (Attenuation)

Answer 15

• No stall at Trp codons
• Leader is fully translated; pauses at STOP codon; blocks region 2
• 3-4 hairpin forms and removes RNA poly.

Question 16

Most stable secondary structure for trp leader mRNA is when…

Answer 16

… regions 1-2 and 3-4 base-pairs and forms hairpin secondary structures

Question 17

Attenuation is only possible because…

Answer 17

transcription and translation are coupled

Question 18

What was the goal of the Lenski experiment?

Answer 18

• To identify mutations that increased fitness (changes in phenotype in long-term evolution)
  
  • Faster gowth rates, increased cell size, increase in DNA repair defects

Question 19

What was used in Lenski experiment? What was transferred?

Answer 19

• (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)

Question 20

What were the results of the Lenski experiment?

Answer 20

• E. coli could catabolize citrate after 33,000 generations; lead to increased density
  
  • Cells evolved way to produce CitT in aerobic conditions

Question 21

Function of CitT transport protein

Answer 21

• 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)

Question 22

Negative control of cit operon (before long-term experiment)

Answer 22

• O₂ present: repressor protein binds to cit promoter & blocks transcription
  
  • No transcription

Question 23

cit operon after duplication event

Answer 23

• cit promoter still inhibited by repressor protein
• Genes and promoters in region rearranged
  
  • repositioned rnk promoter (nucleoside diphosphate kinase regulator) facilitates downstream duplicated copy

Question 24

What can the respositioned rnk promoter do? (after duplication event)

Answer 24

• Transcribe downstream DNA, duplicated copy of citT gene
  
  • O2 present: transmembrane protein made = advantage for transporting energy rich molecules