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???? ??? ?????? ???? N ? C
C
N
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N to C terminal
5 to 3
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CDS
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????????? ????????? ?? RNA ??????
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??????? ???????? ???? ?-N ???? ?-C
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Three-Dimensional tRNA Structure
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• Structure of tRNA
• tRNA coded directly from DNA, single stranded
• Specific sequence of yeast alanine tRNA
• Several bases posttranscriptionally modified
• I insosine, pairs like guanine, wobble base
• Note partial pairing of bases
• Anticodon helps specify selection of alanine as
  the appropriate amino acid

TR10
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tRNA ???? ?????? ??????
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???? 20 ?????? ????? ??? ???? 20 ?????? ?? tRNA
??"? ???? ?. ????? ???? ???? 20 ????? ????? ??
????? ???? tRNA ?????? ??"? ???. ??????
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tRNA

• Aminoacyl-tRNA synthetase (20 ?????)
• one for each amino acid
• NEEDS ENERGY!

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Major Identity Elements in Four tRNAs
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Life cycle of mRNA movie
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64 ?????????? ?-20 ?. ????? ?-3 ??????? ?????????
??? ????????
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Genetic Code

• A codon is made of 3 base pairs
• 64 codons total

1 codon (AUG) encodes methionine and starts
translation of all proteins
3 codons stop protein translation
61 codons encode 20 amino acids (redundant code)
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Cricks Wobble Hypothesis

• mRNA 3rd nucleotide can either be U or C
• tRNA 1st nucleotide guanine (purine) will still
  pair with either mRNA pyrimidine uracil
  or cytosine
• Consequence is single tRNA species will bring
  in same amino acid for both UCC and UCU codons.

TR11
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???????? ?? ?? ????? ???
????? ??? ?-LEU ?? 6 ??????? ????? 4 ??????? ??
CUN
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Wobble role
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Disease-Associated Mutations

• A mutation is a change in the normal base pair
  sequence

Commonly used to define DNA sequence changes that
alter protein function
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Polymorphism
DNA sequence changes that do not alter protein
function (common definition, not technically
correct)
Functional protein
Functional protein
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Single nucleotide polymorphisms (SNP) (in the
coding sequence)
mRNA Protein
Normal
G
C
A
G
Ala
Sequence variant
mRNA Protein
Silent DNA sequence polymorphism
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Polymorphism

• Variation in population
• phenotype
• genotype (DNA sequence polymorphism)
• Variant allele gt 1

Common usage
lt 1
gt 1
Rare or private polymorphism
polymorphism
Normal
??
Disease
disease
Factor V R506Q thrombosis, 3 allele frequency
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Mutations

• Normal
• Missense
• Nonsense
• Frameshift (deletion)
• Frameshift (insertion)

• THE BIG RED DOG RAN OUT.
• THE BIG RAD DOG RAN OUT.
• THE BIG RED.
• THE BRE DDO GRA.
• THE BIG RED ZDO GRA.

Point mutation a change in a single base pair
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Silent Sequence Variants
mRNA Protein
Normal
G
C
A
G
Ala
Sequence variant
mRNA Protein
Sequence variant a base pair change that does
not change the amino acid sequence (a type of
polymorphism)
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Missense Mutations
mRNA Protein
Normal
mRNA Protein
Missense
A
G
C
G
A
C
Ser
Ala
Missense changes to a codon for another amino
acid (can be harmful mutation or neutral
polymorphism)
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Nonsense Mutations
mRNA Protein
Normal
mRNA Protein
Nonsense
Nonsense change from an amino acid codon to a
stop codon, producing a shortened protein
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Frameshift Mutations
mRNA Protein
Normal
mRNA Protein
Frameshift
Frameshift insertion or deletion of base pairs,
producing a stop codon downstream and (usually)
shortened protein
Pre-mature stop codon
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mutation
Missense mutation
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Splice-Site Mutations
Exon 1
Intron
Exon 2
Intron
Exon 3
Exon 2
Exon 3
Exon 1
Altered mRNA
Splice-site mutation a change that results in
altered RNA sequence
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Types of Mutations

• Point Mutations
• Silent
• Missense
• Nonsense
• (frameshift)
• Deletion/Insertion
• small
• large
• Rearrangement

• Transcription
• RNA Processing
• splicing
• poly A
• RNA stability
• Protein level
• processing
• stability
• altered function
• gain
• loss
• new

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eIF3
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eIF3
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The Growing Polypeptide is Linked to an Amino
Acid from an Incoming tRNA

• The C-terminal amino acid of an incomplete
  polypeptide is esterified to a tRNA molecule
• Growing polypeptide is transferred from
  peptidyl-tRNA to incoming aminoacyl-tRNA

P site
A site
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Life cycle of mRNA movie
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Translation Initiation
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Translation Elongation
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Translation Termination
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initiation
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elongation
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termination
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Eukaryotic translation initiation can occur at
IRES
Lodish Fig 4-38
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Chain Termination
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Summary of Chain Termination

• Termination codons recognized by release factors
• Binding of a release factor to a termination
  codon induces hydrolysis of the peptidyl group,
  leading to dissociation of the peptide and the
  uncharged tRNA from the ribosome
• GTP is hydrolyzed release factors, GDP, Pi, and
  mRNA are expelled from the ribosome
• The single release factor eRF recognizes all
  three termination codons in eukaryotes
• GTP hydrolysis accelerates peptide synthesis
• GTP binding factors cause ribosomal components to
  change conformations to facilitate translation
  steps
• Irreversibility of GTP hydrolysis ensures that
  initiation, elongation, and termination will be
  fast and irreversible

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Translational Accuracy, Protein Folding, and
Posttranslational Modifications

• Error rate approximately 1 in 10,000 amino acids
• Protein folding begins before a peptide is fully
  synthesized and is often aided by molecular
  chaperones
• Posttranslational modifications over 150 types
  known
• Initiating fMet often excised
• Proteolysis is often required to activate certain
  proteins or cleave signal sequences
• Phosphorylation
• Glycosylation
• Fatty acylation

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Translation II
Translation
Ada Yonat Movie
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Whos the interpreter?
Transfer RNA (tRNA)

• Brings the amino acid to the right codon
• Unique tRNA for each amino acid
• One side-amino acid
• Other side-anticodon

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IRES (Internal Recognition Site)
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mRNA turnover
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NMD
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RNAi
Life cycle of mRNA movie
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NMD
Exon junction complex (EJC)
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A little terminology

• RNA Interference (RNAi)
• The phenomenon whereby small RNA can induce
  efficient sequence-specific silence of gene
  expression.
• Micro-RNA (miRNA)
• Single-stranded RNAs of 22-nt that are processed
  from 70-nt hairpin RNA precursors by Rnase III
  nuclease Dicer. miRNAs can silence gene activity.
• Small Interfering RNA (siRNA)
• Similar to miRNA, but dsRNA originally found in
  plants, prevent transcription of viral products

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Illustration of miRNA processing
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Differences in miRNA Mode of Action
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miRNA Registry

• http//www.sanger.ac.uk/Software/Rfam/mirna/index.
  shtml
• Latest release contains 4584 predicted and
  verified miRNAs (May 2007)
• 321 predicted and 223 experimentally verified
  in Homo sapiens
• Mouse and human are highly conserved
• Human is not conserved with plants

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???????????? ?????? ?? ???????? ?????? ?????????
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???
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Endoplasmic Reticulum (ER)
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???????? ?-ER
movie
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Endoplasmic Reticulum (ER)
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normal
Prion early
Prion late
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Chaperons
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FD - Familial DysautonomiaRiley-Day Syndrome
Gil Ast
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FD- Familial Dysautonomia

• An autosomal recessive congenital neuropathy
• Poor development and progressive degeneration of
  sensory and autonomic nervous system
• Common in Ashkenazi Jewish population
  carrier 130 (118 in Polish Jews), live births
  13,600
• Symptoms decreased sensitivity to pain and
  temperature, cardiovascular instability,
  gastrointestinal dysfunction, postural
  hypotension, and more
• 50 of patients die before the age of 30

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Major mutation in FD
IKBKAP pre-mRNA
Intron 20
Intron 19
gtaagt
c
Normal splicing
FD mis-splicing
mRNA
IKAP Protein
Normal, 1,332aa, 150kD
Truncated, 714aa, 79kD
The splicing pattern of IKBKAP in FD is tissue
specific
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Examination of splice site quality
IKBKAP acceptor site strength (bits) -2.5
15.8, average 9.47 IKBKAP donor site strength
(bits) 3.2 13.0, average 8.94
Why does this mutation cause aberrant splicing,
particularly in neural tissue?
Figure 1
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