Review for Exam II (Exam this Wed) Bring One of These Multiple

Review for Exam II (Exam this Wed)
Bring One of These
Multiple Choice
• 36 questions (vs. 40 last year)
• Every question worth 2.777 points
• Compared to previous tests:
– Different topics covered – see Study Guide
– lac operon table entries similar to 2014 exam
– Number of “DNA, RNA or protein?” questions
same as 2014 exam
– Only 1 question on eukaryotic gene regulation
• fewer questions overall, should be easier to
complete on time
1
Page-by-page Breakdown
Page
1
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2
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3
4
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Total
# Questions
1
2
4
1
1
2
2
11
5
1
Topic
Bioinformatics
General bacterial genetics
Bacterial sexual processes
Influenza
Chromosome mutations
Chromosome mutations
operons
lac operon table
4 wild-type
5 mutant
2 merozygotes
DNA, protein, or RNA?
Eukaryotic regulation
1
Lactase persistence
4
Mutation
1
Cancer
36 questions (2.7777 points each)
Topic Breakdown
Topic
lac operon
RNA, DNA or protein
Gene Mutations
Bacterial sexual processes
Misc. Bacterial Genetics
Chromosomal Mutations
Eukaryotic Gene Regulation
Genetics of cancer
Bioinformatics
Lactase Persistence
Influenza
# Questions Percent
12
33.3
5
13.9
4
11.1
4
11.1
3
8.3
3
8.3
1
2.8
1
2.8
1
2.8
1
2.8
1
2.8
lac regulation tables are worth 30% of the total!
similar to last year's (below)
2
Protein, DNA, or RNA?
5 questions, 14 %
For each of the following things, indicate whether
it is all or part of a protein, DNA or RNA molecule.
All are taken from the list of terms on the study guide
Gene Mutations
4 questions (11%)
3 out of 4 are on different classifications
of mutations
from those listed on study guide
Bacterial Sexual Processes
4 questions (11%)
What are the characteristics of each?
3
Misc. Bacterial Genetics
3 questions (8%)
• Using media to find mutants (like
questions on quiz)
• trp operon
Chromosomal Mutations
3 questions (8%)
• Emphasis on human chromosomal
mutation
• Medical relevance
• Syndromes caused by chromosomal
mutations
Everything else:
1 questions each - 5 questions (14%)
Emphasis on main points and concepts
(the challenge is figuring out what they are!)
4
Lac Operon
3 levels of expression
– Leaky (repressed, repressor on operator)
– Low (derepressed, no repressor on
operator but no CAP-cAMP either)
– High (derepressed and CAP-cAMP on
promoter)
Confusing Terms
• Inducible does not mean positive
control !
• Repressible does not mean negative
control !
• Example: A gene could be induced by
removal of repressor protein
Sugar source and lac expression –
wild type operon
Lactose
present
Glucose
present
+
+
+
+
-
lac Operon
5
Sugar source and lac expression –
wild type operon
Lactose
present
Glucose
present
lac Operon
-
-
Leaky
repressed
-
+
Leaky
repressed
+
+
Low
derepressed
+
-
Low
Depressed and
CAP-cAMP
Cis and Trans
regulatory sites have cis-acting DNA
sequence elements
cis-acting sites affect promoters on same
DNA molecule
trans-acting factors (proteins) bind to the cisacting sites (e.g. repressors)
trans-acting factors can affect any DNA
molecule in the cell
Lac operon mutants
• function of lac operon was worked out by
studying various mutants
• merozygotes –
– partial diploids of E. coli created by sexduction
with F' plasmid
– F' plasmid can include lac operon – permits study
of combinations of mutant and wild-type elements
• mutant alleles: lacZ -, lacY -, lacA • wild-type alleles: lacZ +, lacY +, lacA +
6
Constitutive mutants
•
•
Phenotype: lac structural genes
transcribed all the time
Two possible mechanisms:
1) lacI - defective repressor can’t bind to
operator
2) lacOC mutant operator can’t bind
repressor
•
How to tell which?
Constitutive mutants
If it’s a lacI - defective repressor a wild-type
lacI+ gene on a plasmid would restore normal
function
If it’s a lacOC mutant operator that can’t bind
repressor a wild-type lacI+ gene on a plasmid
would not restore normal function
Merozygote
F’ Plasmid:
lacI+ O+ lacZ - lacY + lacA –
Mutant Genome: ?? ?? lacZ + lacY – lacA +
If mutant is lacI -, lacI+ on plasmid should
restore normal function
If mutant is lacOC, only genes on plasmid
will be constitutively expressed
7
lacIS superrepressor mutant
• mutant that cannot bind inducer (allolactose)
• repressor is always bound to wild-type operator,
keeps transcription at leaky level
• in a lacI+ / lacIS merozygote, the superrepressor
is dominant because it binds to any lac operator
(trans-acting)
lacIS superrepressor mutant and a
OC constitutive operator together
• lacIS product doesn’t bind inducer (allolactose),
but still needs a wild-type operator to bind to
• An OC operator will not bind any repressor,
doesn’t matter if it’s lacI or lacIS
lacP - promoter defects
• mutation in -10 or -35 region of the lac
promoter
• RNA polymerase cannot bind to promoter
• In lacP - / lacP + merozygote, the promoters
are cis-acting, and don’t affect each other
8
From 2014 Exam
From 2014 Exam
From 2014 Exam
9
From 2013 Exam
From 2013 Exam
From 2013 Exam
10
Mutants (from 2012)
Mutants (from 2011exam)
Merozygotes
(from 2012 Exam)
11
Merozygotes
(from 2011Exam)
trp operon - Repression
• five structural genes encode enzymes for
synthesis of amino acid tryptophan
• promoter (trpP), operator (trpO) and
regulatory gene (trpR)
• product of trpR is repressor, TrpR
• TrpR binds tryptophan (corepressor)
• tryptophan abundant: TrpR-tryptophan binds
to operator and represses transcription
• tryptophan scarce: tryptophan leaves TrpR,
TrpR leaves operator, operon is derepressed
trp operon - Attenuation
• attenuator region between operator and
first structural gene (trpE)
• mRNA transcribed from attenuator
(leader transcript) has four subregions
that form 3 different stem-loop
structures
• if stem-loops with regions 1-2 and 3-4
form, stem-loops with regions 2-3 can’t
form
12
terminator
trp operon leader peptide
• leader transcript also contains a short
peptide-encoding gene with two
adjacent tryptophan codons
• region 1 is located within the leader
transcript
trp Operon
13
Attenuation of trp
Abundant tryptophan:
• ribosome translates leader
peptide quickly, position of
ribosome causes region 3-4
stem-loop to form
• Region 3-4 stem-loop structure
acts as a terminator, stops
transcription
Attenuation of trp
Low tryptophan:
• ribosome pauses on tryptophan
codons in leader sequence and
prevents region 1 from pairing with
region 2
• region 2-3 stem-loop forms
instead, which prevents region 3-4
stem loop (the terminator) from
forming
• no termination, transcription
continues
14