Which of the following is a true statement regarding the transfer of information from DNA to RNA.
1. It is always irreversible
2. It occurs during a process known as translation
3. It is sometimes reversible
a) 1
b) 2
c) 3
d) 1 and 2
e) 2 and 3
Consider the DNA template 3'—AAATTTAGCCA—5'. When transcribed, which of the following
is the correct resulting transcript?
a) 5'—TTTAAATCGGT—3'
b) 5'—UUUAAAUGCCA—3'
c) 5'—UUUAAAUCGGU—3'
d) 3'—TTTAAATCGGT—5'
e) 3'—AAATTTTAGCCA—5'
mRNA strands that will specify amino acids in the protein gene product are also known as:
a) Coding strands
b) Sense strands
c) Antisense strands
d) Coding strands and Sense strands
e) Coding strands and Antisense strands
Which of the following is the RNA polymerase that is used for transcription in eukaryotes
1. RNA Polymerase 1
2. RNA Polymerase 2
3. RNA Polymerase 3
a) 1
b) 2
c) 3
d) 1 and 2
e) All of these
Consider the DNA template 3'—CAATTTAGCCA—5'. When translated, which of the following
is the correct resulting transcript?
a) 5'—TTTAAATCGGT—3'
b) 5'—UUUAAAUGCCA—3'
c) 5'—GUUAAAUCGGU—3'
d) 3'—TTTAAATCGGT—5'
e) 3'—AAATTTTAGCCA—5’
f) None of them
Glucose Regulation of β-pancreatic Cells
(Blood)
Sugars
Blood
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RECEPTOR
Activation ?
AP1
Modes of regulation vs.DNA-binding domain
Transcription factors are activated in different ways
Regulation
DNA-binding
Most regulatory schemes
involve a modification of
a protein that changes its
conformation or location.
Activators may have the
following domain/motifs:
Steroid receptor
Zinc finger
Helix-turn-helix
Helix-loop-helix
Leucine zipper
Sub-cellular Localization?
Chapter 12
Translation and the Genetic
Code
© John Wiley & Sons, Inc.
Chapter Outline
Protein Structure
Protein Synthesis: Translation
The Genetic Code
Codon-tRNA Interactions
© John Wiley & Sons, Inc.
Protein Structure
Proteins are macromolecules
composed of 20 different amino
acids.
Selenocysteine
Pyrrolysine
3- and 1-Letter
Sec U
Pyl O
© John Wiley & Sons, Inc.
Amino Acids
 Proteins are made of peptides (~10 to15% of total mass)
 A polypeptide is a long chain of amino acids.
 Amino acids have a free amino group, a free carboxyl
group, and a side group (R).
 R group is responsible for structural and functional
diversity.
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© John Wiley & Sons, Inc.
Peptide Bonds
Amino acids are joined by peptide bonds.
The carboxyl group of one amino acid is
covalently attached to the amino group of the
next amino acid.
It is directional
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Levels of Protein Structure
Sequence
spatial
3-D
association//function
function
-sheet or
-strand
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Protein Folding: α-helix
Peptide bond backbone
R groups jut out
Every peptide bond – H bonded
3 amino acids separate per turn
H bonds nearly parallel to the main axis
Protein Folding: β-sheet (strand)
R groups jut out at alternate sides
All amino & carbonyl are in - H bonded
Intra- or inter- molecular
Parallel or anti-parallel
Molecular Interactions Determining
Tertiary Structure
Covalent interaction:
strong
join monomers
stabilize 3D structure
(disulfide bonds)
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Non-covalent interaction: equally important but Weak
Hydrogen bondsbetween a hydrogen atom in a polar covalent
bond & a second electronegative atom
Ionic bondselectrostatic interaction two oppositely charged ions
van der Waals interactionsinteractions between dipoles (requires close
proximity and specific orientation)
Hydrophobic interactionstendency of nonpolar groups to associate with each other
 Most genes exert their effect(s) on the phenotype of an
organism through proteins, which are large macromolecules
composed of polypeptides.
 Each polypeptide is a chain-like polymer assembled from
different amino acids.
 The amino acid sequence of each polypeptide is specified by
the nucleotide sequence of a gene.
 The vast functional diversity of proteins results in part from
their complex three-dimensional structures.
© John Wiley & Sons, Inc.
One Gene-One Colinear Polypeptide
The sequence of nucleotide pairs specifies a
colinear (linear) sequence of amino acids in its
polypeptide product
Gene
polypeptides
© John Wiley & Sons, Inc.
Drosophila: Genes required for eye pigments (ENZYMES)
Mold: Neurospora Crassa
© John Wiley & Sons, Inc.
Salts
Sugar
Biotin
© John Wiley & Sons, Inc.
One gene--pathway--one enzyme//one polypeptide
© John Wiley & Sons, Inc.
Coding seq. and polypeptide
Tryptophan synthetase: enzymatic activity
trpA and TrpB genes
© John Wiley & Sons, Inc.
Beadle and Tatum's Experiments with
Neurospora led to the one gene-one enzyme
hypothesis, which was subsequently modified to
the one gene-one polypeptide concept.
The sequences of nucleotide pairs in a gene and
amino acids in its polypeptide product are
colinear.
© John Wiley & Sons, Inc.
Protein Synthesis: Translation
The genetic information in mRNA
(nucleotides) molecules is translated into
the amino acid sequences of
polypeptides according to the
specifications of the genetic code.
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
The Macromolecules of Translation
mRNA as one molecule
rRNA molecules (3-5) of each ribosome
tRNA Molecules (40-60)
Soluble proteins involved in polypeptide chain
initiation, elongation, and termination
© John Wiley & Sons, Inc.
Polypeptides….. to be produced in …
------free cytosolic ribosones (polyribosomes)
-----ribosomes associated to endoplasmic reticulum/Golgi/vesicles
Signal sequence:
secreted
Membrane associated
Ribosomes
© John Wiley & Sons, Inc.
Synthesis and Processing of the 30S
rRNA Precursor in E. coli
© John Wiley & Sons, Inc.
Synthesis and Processing of the
45S rRNA Precursor in Mammals
© John Wiley & Sons, Inc.
rRNA Genes
rRNA Genes in E. coli
– Seven rRNA genes distributed among three sites
on the chromosome
rRNA Genes in Eukaryotes
– rRNA genes are present in hundreds to thousands
of copies
– The 5.8S-18S-28S rRNA genes are present in
tandem arrays in the nucleolar organizer regions
of the chromosomes.
– The 5S rRNA genes are not nucleolar and they
are distributed over several chromosomes.
© John Wiley & Sons, Inc.
Transfer RNAs (tRNAs)
 tRNAs are adapters
between amino acids and
the mRNA molecules.
 The amino acid is
covalently attached to the
3’ end of the tRNA.
 tRNAs often contain
modified nucleosides.
 tRNAs have
complementary sequence
to mRNA. The anticodon
of the tRNA base pairs
with the codon of mRNA.
© John Wiley & Sons, Inc.
tRNA Structure
© John Wiley & Sons, Inc.
Specificity of tRNAs
tRNA molecules must have the correct
anticodon sequence.
tRNA molecules must be recognized by the
correct aminoacyl-tRNA synthetase.
tRNA
aa-tRNA
tRNA molecules must bind to the
appropriate sites on the ribosomes.
© John Wiley & Sons, Inc.
tRNA Binding Sites on the
Ribosome
specificity
© John Wiley & Sons, Inc.
Stages of Translation
Chain Initiation: they are events
before the formation of a di-peptide
Chain Elongation
Chain Termination
© John Wiley & Sons, Inc.
Translation Initiation in E. coli
30S subunit of the ribosome
 Special Initiator tRNA (tRNAfMet), UAC
mRNA (AUG)
Initiation Factors IF-1, IF-2, and IF-3
One molecule of GTP (Energy)
50S subunit of the ribosome
© John Wiley & Sons, Inc.
Amino group is blocked
Interaction between mRNA
and 16S rRNA
© John Wiley & Sons, Inc.
The Shine-Dalgarno
Sequence
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
Translation Initiation in Eukaryotes
The amino group of the methionine on the initiator
tRNA is not formylated (~linked to the CAP binding
protein)
The initiation complex forms at the 5’ terminus of the
mRNA, not at the Shine-Dalgarno/AUG translation
start site.
The initiation complex scans the mRNA for an AUG
initiation codon. Translation usually begins at the first
AUG.
Kozak’s Rules describe the optimal sequence for
efficient translation initiation in eukaryotes (5’GCC(AorG)CCAUGG-3’)
© John Wiley & Sons, Inc.
tRNAimet
Polypeptide Chain Elongation
An aminoacyl-tRNA binds to the A site of the
ribosome.
The growing polypeptide chain is transferred from
the tRNA in the P site to the tRNA in the A site by the
formation of a new peptide bond.
The ribosome translocates along the mRNA to
position the next codon in the A site (5’ to 3’). At the
same time,
– The nascent polypeptide-tRNA is translocated from the A
site to the P site.
– The uncharged tRNA is translocated from the P site to the E
site.
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
Guanine Exchange Factor,
GTP activating Protein
GEF
GDP
Inactive
GAP
Rate of hydrolysis
Pi+
GTP
Time (sec)
active
Guanine Exchange Factor,
GTP
GTP activating Protein
GEF
GDP
GTP
GDP
GAP
Rate of Exchange
Inactive
Time (sec)
active
Exchange
Hydrolysis
GEF
Elongation factor
© John Wiley & Sons, Inc.
Ribosome Recycling Factor (RRF)
23S rRNA
+RRF
© John Wiley & Sons, Inc.
EF-Tu ?
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
Elongation of Fibroin Polypeptides
© John Wiley & Sons, Inc.
Polypeptide Chain Termination
 Polypeptide chain termination occurs when a chain-termination
codon (stop codon) enters the A site of the ribosome.
 The stop codons are UAA, UAG, and UGA.
 When a stop codon is encountered, a release factor (Protein)
binds to the A site.
----RF1: UAA, UAG.
----RF2: UAA, UGA.
A water molecule is added to the carboxyl terminus of the
nascent polypeptide, causing termination.
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
 Genetic information carried in the sequences of nucleotides in
mRNA molecules is translated into sequences of amino acids
in polypeptide gene products by intricate macromolecular
machines called ribosomes.
 The translation process is complex, requiring the participation
of many different RNA and protein molecules.
 Transfer RNA (tRNA) molecules serve as adaptors, mediating
the interaction between amino acids and codons in mRNA.
 The process of translation involves the initiation, elongation,
and termination of polypeptide chains and is governed by the
specifications of the genetic code.
© John Wiley & Sons, Inc.
The Genetic Code
The genetic code is a non-overlapping
sequence, with each amino acid plus
polypeptide initiation and termination
specified by RNA codons composed of
three nucleotides.
© John Wiley & Sons, Inc.
Properties of the Genetic Code (~1960)
 The genetic code is composed of nucleotide triplets.
 The genetic code is
--non-overlapping.
--comma-free.
--degenerate.
--ordered.
--organized by start and stop codons.
--nearly universal.
© John Wiley & Sons, Inc.
A Triplet Code
and the “Open” Reading
Frame
Bacteriophage T4 ( F. Crick)
is a portion of a DNA molecule that, when translated into amino acids, contains no stop codons
and so can potentially translate as a polypeptide chain.
Fragment of DNA that does not contain a nucleotide triplet which stops translation
before formation of a complete polypeptide
© John Wiley & Sons, Inc.
A Single-Base Pair Insertion
Alters the Reading Frame
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A suppressor mutation restores
the original reading frame.
?
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Insertion of 3 base pairs does not
change the reading frame.
© John Wiley & Sons, Inc.
Evidence of a Triplet Code: In Vitro
Translation Studies
Trinucleotides were sufficient to stimulate specific
binding of aminoacyl-tRNAs to ribosomes.
Chemically synthesized (in vitro) mRNAs containing
repeated dinucleotide (UG) sequences directed the
synthesis of copolymers with alternating amino acid
sequences.
mRNAs with repeating trinucleotide (UUG/UUU)
sequences directed the synthesis of a mixture of three
homopolymers.
© John Wiley & Sons, Inc.
Deciphering the Genetic Code
4 nucleotides vs triplets for amino acids…..43= 64
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
The Genetic Code
Initiation and termination Codons
– Initiation codon: AUG
– Termination codons: UAA, UAG, UGA
Degeneracy:
– Partial: UUU, UUC (Phe,F) ; UUA, UUG (Leu, L)
– Complete:GUU, GUC, GUA and GUG (Val, V)
Ordered
Nearly Universal (exceptions: mitochondria; UGA=Trp
W... Stop codon) © John Wiley & Sons, Inc.
 Each of the 20 amino acids in proteins is specified by one or
more nucleotide triplets in mRNA.
 Of the 64 possible triplets, given the four bases in mRNA, 61
specify amino acids and 3 signal chain termination.
 The code is “non-overlapping”, with each nucleotide part of a
single codon,
 “Degenerate”, with most amino acids specified by two to four
codons,
 and “ordered”, with similar amino acids specified by related
codons.
 The genetic code is nearly universal; with minor exceptions,
the 64 triplets have the ©same
meaning
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& Sons, Inc. in all organisms.
Overlapping
Codon
Non-Overlapping
AUU UUG UGC
AUU GCU GAG GUU GAC
AUU GCU GAG
Codon-tRNA Interactions
Codons in mRNA molecules are
recognized by amino acyl-tRNAs
during translation.
Different tRNAs recognize different codons on mRNA for
any amino acid
Anticodon tRNAs recognize different codons on mRNA
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
Wobble (wiggle) hypothesis:
--base pairing AU and CG
----first two bp is very strict
----Third bp is not strict
© John Wiley & Sons, Inc.
inosine
Base-Pairing with Inosine at the
Wobble Position (by Crick)
© John Wiley & Sons, Inc.