???? ?????? ?????? ??????? ?????

1
(No Transcript)
2
????????
???? ?????? ?????? ??????? ????? ?????? ????????
3
????? ????
????? ????
4
???? ????? ??????
????? ??
5
???? ??? ?????? ???? N ? C
C
N
6
N to C terminal
5 to 3
7
CDS
8
????????? ????????? ?? RNA ??????
9
???????? RNA ?????? ???? ??????
10
(No Transcript)
11
(No Transcript)
12
??????? ???????? ???? ?-N ???? ?-C
13
????-?????
14
Three-Dimensional tRNA Structure
15

• 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
16
tRNA ???? ?????? ??????
17
(No Transcript)
18
(No Transcript)
19
???? 20 ?????? ????? ??? ???? 20 ?????? ?? tRNA
??"? ???? ?. ????? ???? ???? 20 ????? ????? ??
????? ???? tRNA ?????? ??"? ???. ??????
20
(No Transcript)
21
(No Transcript)
22
tRNA

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

23
Major Identity Elements in Four tRNAs
24
(No Transcript)
25
(No Transcript)
26
(No Transcript)
27
Life cycle of mRNA movie
28
(No Transcript)
29
(No Transcript)
30
64 ?????????? ?-20 ?. ????? ?-3 ??????? ?????????
31
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)
32
???? ?????
33
????? ?????????-?????
34
(No Transcript)
35
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
36
??? ????????
????? ????
???????? ?? ?? ????? ???
????? ??? ?-LEU ?? 6 ??????? ????? 4 ??????? ??
CUN
?????
37
????? ????????? ????? ?????? ???????? ?? ???
?-tRNA ??????? (Leu) ?? ??? ???? ?-wobble ?? C.
??? ????? ????? ?????? ??? ??? ?????

• 1. ??
• 2. ??
• 3. ?? ???? ??? ?? ??? ?? ?????
• ?? G ?????
• 4. ??, ?? ?????????? ??????? ????? ????? ???????.

38
Wobble role
39
??????? ??? ???
40
(No Transcript)
41
????????????
42
(No Transcript)
43
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
44
Polymorphism
DNA sequence changes that do not alter protein
function (common definition, not technically
correct)
Functional protein
Functional protein
45
Single nucleotide polymorphisms (SNP) (in the
coding sequence)
mRNA Protein
Normal
G
C
A
G
Ala
Sequence variant
mRNA Protein
Silent DNA sequence polymorphism
???????? ???-????????
46
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
47
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
48
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)
49
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)
50
Nonsense Mutations
mRNA Protein
Normal
mRNA Protein
Nonsense
Nonsense change from an amino acid codon to a
stop codon, producing a shortened protein
51
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
52
????? ?????
53
mutation
Missense mutation
54
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
55
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

56
Translation II
????? ??? ?? ?. ????? ????? ??? ??? ?? ????
?? ?. ????? ??? ?????? ????????
57
(No Transcript)
58
(No Transcript)
59
(No Transcript)
60
(No Transcript)
61
(No Transcript)
62
(No Transcript)
63
(No Transcript)
64
eIF3
65
eIF3
66
??????? ?????????
67
Translation
68
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
69
(No Transcript)
70
(No Transcript)
71
(No Transcript)
72
??? ????????
Life cycle of mRNA movie
73
(No Transcript)
74
(No Transcript)
75
(No Transcript)
76
Translation Initiation
77
Translation Elongation
78
Translation Termination
79
initiation
80
elongation
81
termination
82
Eukaryotic translation initiation can occur at
IRES
Lodish Fig 4-38
83
Chain Termination
84
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

85
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

86
Ada Yonat Movie
87
(No Transcript)
88
(No Transcript)
89
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

90
(No Transcript)
91
(No Transcript)
92
(No Transcript)
93
???????
94
???????? ???????????
95
IRES (Internal Recognition Site)
96
mRNA turnover
97
NMD
98
RNAi
Life cycle of mRNA movie
99
NMD
Exon junction complex (EJC)
100
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

101
microRNAs

• MicroRNAs (miRNAs) are noncoding RNAs of 22
  nucleotides that induce post-transcriptional gene
  silencing through base-pairing with their target
  mRNAs.
• More than 700 miRNAs have been identified in
  human
• Each miRNA can regulate hundreds of different
  mRNA molecules
• MicroRNAs (miRNAs) play important roles in a
  broad range of biological processes including
  development, cellular differentiation,
  proliferation, and apoptosis

102
Illustration of miRNA processing
103
miRNA processing
104
Differences in miRNA Mode of Action
105
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

???? ?? ??????? ???????? ?????
106
(No Transcript)
107
(No Transcript)
108
(No Transcript)
109
????????? ????? ?????? ????? ??????? ????? ???????
110
(No Transcript)
111
???????????? ?????? ?? ???????? ?????? ?????????
?????? ??? ??-???????????
???
112
Endoplasmic Reticulum (ER)
113
???????? ?-ER
movie
114
(No Transcript)
115
(No Transcript)
116
(No Transcript)
117
(No Transcript)
118
Endoplasmic Reticulum (ER)
119
???????????? ????? ??????
120
???????????
121
(No Transcript)
122
(No Transcript)
123
(No Transcript)
124
(No Transcript)
125
????????- ????? ????? ?????? ?????
126
normal
Prion early
Prion late
127
(No Transcript)
128
Chaperons
??????? ??????? ??????? ?????? ???? ?? ??????
????? ????
129
(No Transcript)
130
????????? ????? ????? ?? ??????? ???
?????????
?????? ?????? ????? ?? ?? ??? ????
131
???????
132
FD - Familial DysautonomiaRiley-Day Syndrome
Gil Ast
133
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

134
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
135
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
136
(No Transcript)