Transcript Document

Please feel free to chat amongst yourselves
until we begin at the top of the hour.
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Seminar Agenda
 Review of Course Information
 Seminar Discussion Questions
 Questions & Answers
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Review of Course Information:
Mid-Term Exam
 This Exam is due by the end of Unit 5 of the course.
 Worth 100 points.
 The exam will cover the materials in Units 1-5; it includes
everything that was covered in the required readings, lessons,
activities, Seminar, and discussion boards. This Exam may
only be submitted once and must be taken in one sitting; you
may not log-out and log-back in.
 Exams must be taken during the Unit week (Wednesday to
Tuesday). There will be no extensions.
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Any questions?
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Discussion Questions 1
 If DNA consisted of only two nucleotides
(say G and C) in any sequence, what is the
minimum number of adjacent nucleotides
that would be needed to specify uniquely
each of the 20 amino acids?
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Genetic Code
• The genetic code is the list of all codons and the amino
acids that they encode
• Main features of the genetic code were proved in genetic
experiments carried out by F.Crick and collaborators:
• Translation starts from a fixed point
• There is a single reading frame maintained throughout
the process of translation
• Each codon consists of three nucleotides
• Code is nonoverlapping
• Code is degenerate: each amino acid is specified by
more than one codon
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Fig. 8.24
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Genetic Code
• Most of the codons were
determined from in
vitropolypeptide
synthesis
• Genetic code is universal
= the same triplet codons
specify the same amino
acids in all species
• Mutations occur when
changes in codons alter
amino acids in proteins
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Discussion Questions 1
 If DNA consisted of only two nucleotides
(say G and C) in any sequence, what is the
minimum number of adjacent nucleotides
that would be needed to specify uniquely
each of the 20 amino acids?
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Discussion Questions 1
 If DNA consisted of only two nucleotides (say G and C) in
any sequence, what is the minimum number of adjacent
nucleotides that would be needed to specify uniquely each
of the 20 amino acids?
 Answer: The requirement is that 2n>20, or n=5. If
the codons were of four nucleotides, then only 16
amino acids could be specified because 24=16. A
codon of five nucleotides does the job because
25=32. In fact, it leaves some extra codons for stop
codons and redundancy.
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Discussion Questions 2
 Poly-U RNA codes for polyphenylalanine. If
a G is added to the 5’ end of the molecule,
the polyphenylalanine has a different amino
acid at the amino terminus, and if a G is
added to the 3’ end, there is a different
amino acid at the carboxyl terminus. What
are the amino acids?
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Discussion Questions 2
 Poly-U RNA codes for polyphenylalanine. If a G is added to the 5’ end of
the molecule, the polyphenylalanine has a different amino acid at the
amino terminus, and if a G is added to the 3’ end, there is a different amino
acid at the carboxyl terminus. What are the amino acids?
Answer:
-> NH3-Val-Phe-Phe-Phe-Leu-COOH
 Codons are read from the 5’ end of the mRNA. The first
amino acid in the polypepetide chain is that at amino end, and
the last is at the carboxy end. Therefore, a G added to the
5’end of poly-U results in the codon GUU as the first codon in
the RNA and hence Valine (val as the first amino acid in the
polypeptide. When G is added to the 3’end, it results in a UUG
codon, which codes for a leucine (leu) at the carboxyl terminus
of the polypeptide.
 5’-GUU UUU UUU UUU UUG-3’
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Discussion Questions 3
 Why are mutations of the lac operator often
called a cis-dominant? Why are some
constitutive mutations of the lac repressor
(lacI) called trans-recessive? Can you think
of a way in which a noninducible mutation
in the lacI gene might be trans-dominant?
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Operon Model of Regulation
• In bacterial systems, when several enzymes act
in sequence in a single metabolic pathway,
usually either all or none of the enzymes are
produced
• This coordinate regulation results from control of
the synthesis of one or more mRNA molecules
that are polycistronic
• Polycistronic mRNAs are transcribed from an
operon: a collection of adjacent structural genes
regulated by an operator
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lac Operon
• The genetic regulatory mechanism in bacteria was
first explained by the operon model of François
Jacob and Jacques Monod
• They studied lactose-utilization system in E.coli
• The lactose-utilization system consists of two kinds
of components: structural genes (lacZ and lacY),
which encode proteins needed for the transport and
metabolism of lactose, and regulatory elements (the
repressor gene lacI, the promoter lacP, and the
operator lacO)
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lac Operon
• The products of the lacZ (enzyme b-galactosidase)
and lacY (transporter lactose permease) genes are
coded by a single polycistronic mRNA
• The linked structural genes, together with lacP and
lacO, constitute the lac operon
• The promoter mutations (lacP- ) eliminate the ability
to synthesize lac mRNA
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lac Operon
• The product of the lacI gene is a repressor, which
binds to a unique sequence of DNA bases
constituting the operator
• When the repressor is bound to the operator,
initiation of transcription of lac mRNA by RNA
polymerase is prevented
• Because the repressor is necessary to shut off
mRNA synthesis, regulation by the repressor is
negative regulation
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lac Operon
• Lactose (inducer) stimulate mRNA synthesis by
binding to and inactivating the repressor
Fig. 9.4a, b
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lac Operon
• In the presence of an
inducer, the operator
is not bound with the
repressor, and the
promoter is
available for the
initiation of mRNA
synthesis
Fig. 9.4c
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Discussion Questions 3
 Why are mutations of the lac operator often called a cis-
dominant?
X
 Answer: Mutations of operators are called cis-dominant
because, to exhibit their effect, they must be present in the
same DNA molecule as the gene they control (that is, the
regulatory sequence and the gene must be in a cis
configuration). Because the phenotypic effects are
observed in partial heterozygotes carrying a normal
operator on the other genetic element (for example in an
F’lac plasmid), the mutations are said to be cis-dominant.) 20
Discussion Questions 3
 Why are some constitutive mutations of the lac
repressor (lacI) called trans-recessive?
X
 Answer: Constitutive mutations in lacI are called
trans-recessive because their effect is overridden
by a copy of lacI+ anywhere in the genome (that is
they are in a trans configuration relative to the
mutant allele).
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Discussion Questions 3
 Can you think of a way in which a noninducible mutation
in the lacI gene might be trans-dominant?
 Answer: One can imagine mutations of lacI that bind to
the operator irrespective of whether an inducer is present.
Such a mutant protein would repress all lac operons, even
if normal repressor molecules were present. Such
mutations would, therefore, be dominant to the normal
repressor. Mutants of this type are actually known: They
are call “superrepressor” mutants and are designated lacIs.
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Any questions?
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