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Week of MAR-14

M T W R F
10-11 10-11
Iryna Run
office hours office hours
LLAB 114 LLAB 114
11:00-11:50 noon-1:00 11:00-11:50 11:00-11:50
Class GG Class Class
LLAB 10 office hours LLAB 10 LLAB 10
Questions? LL114
LLAB 319
12-1
Steve
office hours
LLAB 114
5-6:30 6:30-8:00 6:30-8:30 5:15-6:15
Review Review Exam-2 GG
LLAB 10 LLAB 10 LLAB 10 office hours
LLAB 319

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221.S16
What is on Exam-2?
Content
• The primary emphasis for the exam will be the material that we covered in
class from Monday 2/8/16 (Allele interactions) through Wednesday 3/2/16
(RNA splicing).

• You are also responsible for the corresponding reading in the text (see
syllabus) and for the required reading listed on our Canvas site.

• You should also have a good understanding of the material & problems
corresponding to the prerequisites for the course (see syllabus).

• As you might imagine, you should be pretty facile at solving problems like
those included on the syllabus and in the recitations.

Review sessions
• Monday 3/14 5:00-6:30 LLAB 10
• Tuesday 3/15 6:30-8:00 LLAB 10

Exam-1
• Wednesday 3/16 6:30-8:30 LLAB 10
• Simple calculators are permitted for this exam. Laptops, iPads, PDAs,
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smartphones, etc. are NOT permitted
221.S16
Lac operon

constitutive cistron (lac I)

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221.S16
Lac operon

polycistronic mRNA

allolactose

coordinate regulation
•transcription inducible operon
•translation

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221.S16
Bacterial gene regulation

Cistron
Any sequence of nucleotides in a genome that encodes a functional RNA (a gene)
plus adjacent transcriptional control DNA elements

Operon
A set of adjacent structural genes,
transcribed as a polycistronic mRNA,
plus the adjacent transcriptional control DNA elements

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221.S16
E. coli Trp operon

E. coli

trpEDCBA

attenuator

tryptophan

Feedback
inhibition
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221.S16
E. coli Trp operon
trpR
P

trp repressor (inactive; binds DNA with high off rate)

tryptophan (co-repressor)

[tryptophan + trp repressor] complex (active; binds DNA with low off rate)

trpE…

-40 +20 P1 : promoter (RNAP binding site)


-20 +1 o : operator (repressor binding sites)

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221.S16
E. coli trpR regulon

trpR
trpR trpEDCBA
repressor aroH
protein
aroL
mtr

Regulon
Genes that are transcribed in a manner that is coordinated by the
same regulatory protein (e.g., trpR)

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221.S16
E. coli trpEDCBA operon

TrpR binding site

5’-TATCGTACTCTTTAGCGAGTACAACC-3’
3’-ATCGCATGAGAAATCGCTCATGTTGG-5’

DNA palindrome: both strands have the same nucleotide sequence but in
antiparallel orientation.

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221.S16 Yang et al. (1996) J. Mol. Biol. 258, 37–52
E. coli trpEDCBA operon

TrpR binding site

5’-TATCGTACTCTTTAGCGAGTACAACC-3’
3’-ATCGCATGAGAAATCGCTCATGTTGG-5’

TrpR homodimer

DNA

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221.S16 Strickland et al. (2008) PNAS 105:10709-10714
E. coli trpEDCBA operon

TrpR binding site

5’-TATCGTACTCTTTAGCGAGTACAACC-3’
3’-ATCGCATGAGAAATCGCTCATGTTGG-5’

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221.S16 Yanofsky (2007) RNA 13:1141-1154
E. coli Trp operon

a. How do you know tryptophan is important in Trp operon regulation?

genotype tryptophan trpB mRNA


(in growth medium) (arbitrary units)

trpR+ - 7000

trpR+ + 10

700-fold repression

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221.S16
E. coli Trp operon

leader region

+1 162

trpE…
R
27 68 110 141

60-83 110-134 stem-loops


1/2 3/4

leader peptide (14 aa) attenuator

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221.S16
E. coli Trp operon

b. How much repression is due to trpR? repression


c. How much repression is due to trp a (attenuator)? attenuation

genotype tryptophan trpB mRNA


(in growth medium) (arbitrary units)

trpR+ trp a+ + 10 70-fold repression by trpR

trpR+ trp a + 100


trpR- trp a+ + 700 10-fold repression by
trp attenuator sequence
trpR- trp a + 7000

trp a+ : trp attenuator Attenuation not dependent on trpR

trp a : deletion of trp attenuator

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221.S16
E. coli Trp operon

leader region

+1 162

trpE…
R

How would you demonstrate that trpR works in trans?

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221.S16
Making partial diploids in bacteria
plasmid
An autonomously replicating
extrachromosomal DNA molecule

episome
A plasmid able to integrate into the host
chromosome

F factor (fertility factor)


A bacterial episome that confers
chromosome donor ability

F’ factor
An F factor that carries a part of the
bacterial chromosome
e.g., F’lac Faulty outlooping produces F´, an F plasmid that contains
chromosomal DNA

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221.S16
Characterization of mutations: lacI- (loss of function)

all strains are O+

lacI- leads to constitutive activity of the structural genes

lacI+ is dominant to lacI-

lacI+ is trans-acting

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221.S16
E. coli Trp operon

leader region

+1 162

trpE…
R

How would you demonstrate that trpR works in trans?

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221.S16
E. coli Trp operon

leader region

+1 162

trpE…
R
27 68 110 141

60-83 110-134 stem-loops


1/2 3/4

leader peptide (14 aa) attenuator

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221.S16
E. coli Trp operon: leader region

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221.S16
E. coli Trp operon: alternative stem-loop structures

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221.S16 Hartwell et al. (2008)
E. coli Trp operon

tryptophan present

3/4

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221.S16 Hartwell et al. (2008)
E. coli Trp operon

tryptophan absent
2/3

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221.S16 Hartwell et al. (2008)