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AGR2451 Lecture 3 (M. Raizada)
•Questionnaire -useful
•This week’s reading on reserve:
Plants, Genes and Agriculture, Ch.9 (p.240-262)
•10 minute chat sign-up sheet
•Review of last lecture (did you read your notes?)
“Proteins and Protein Folding”
1. Because of the variety of amino acids available,
evolution selected proteins to be the main enzymes of life.
2. Enzymes increase the probability that two reactive
molecules will form or break a bond at an active site.
3. Local amino acid charges interact with nucleotides,
other amino acids, chemicals very precisely. Any change
in the local charge or size can cause changes in protein
conformation or binding.
4. The addition or loss of small molecules (phosphates,
lipids, glucose) can be used as an “on/off” switch for
protein activity.
5. Proteins are basically a carbon scaffold upon which
charged or hydrophobic surfaces exist to do biochemistry.
6. Proteins do NOT carry the genetic code, but must
interact with the genetic code.
Slide 3.1
Proteins carry out the primary work in a cell, but the set of instructions
to make the proteins (the instruction book of life) cannot be encoded by
amino acids. Instead,evolution chose nucleotides(DNA, RNA). Why?
What are the two requirements of the instruction book of life?
1. The molecular book must be able to instruct amino acids
to join into a long chain with ~100% accuracy.
2. The code for making proteins must be inherited, and with ~100%
accuracy. To allow an organism to grow >1 cell, or to have
multiple progeny, the code must be able to duplicate.
DNA (or RNA) meets these requirements: How?
The key is ~100% reliable hydrogen-bonding between the
nucleotides A-T(U) and G-C.
Hydrogen bonding permits two activities of DNA:
-one strand of DNA is adequate to instruct amino acid joining.
-two strands allow the code to be duplicated and inherited with ~100%
accuracy.
From Chapter 11 Intro to Genetic Analysis
Slide 3.2
Chemical Features of DNA
From Chapter 11 Intro to Genetic Analysis
The structure consists of carbon rings(backbone of 5 carbon sugars),
nitrogen-containing “bases” to permit hydrogen-bonding between
A-T(U) and G-C, all connected by phosphate/oxygen bonds
(phosphodiester linkages).
Polar molecules (N, O) allow for hydrogen bond base-pairs which is
key to double strandedness which is essential to replicate life.
The atoms chosen, C, N, O, P were major constituents of Earth.
Plants (and all of life), but obtain usable forms of these atoms.
Slide 3.3
Inheritance of the Instruction Book of Life (DNA)
•Need 1 DNA strand for instructions, but 2 strands for
inheritance. Why?
•Because of H-bonding, each strand can act as a
mirror-image template of the opposite strand, to
permit DNA replication
•the double helix must unwind using enzymes and
replicate using enzyme DNA Polymerase to join
nucleotides together in a long chain using A-T, G-C
hydrogen bonding as the set of instructions
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P.322 Griffiths
Introduction to Genetic Analysis (6th ed)
Chapter 11 A.J. Griffiths et al.
WH Freeman and Co. Publishers, NY, 1996
Slide 3.4
One strand of nucleic acid (mRNA) is the
organizer of protein formation
•codon - 3 bp RNA sequence that encodes
an amino acid
•basis of genetic code
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•tRNA binds an amino acid and
carries it to the mRNA template, where
H-bonding between 3 letters of tRNA
and mRNA allow correct placement
of amino acid in the growing
amino acid chain
•3 positions needed for code, because
20 different amino acids. A two
Position code only has 16 combo’s
given 4 possible letters.
•the amino acid polymer is
built inside the Ribosome
complex consisting ofmRNAs, tRNAs,
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•”Translation” - conversion
of 4 base genetic code to
20 amino acid protein code
•What was the key invention by
evolution to allow DNA/RNA to
organize protein formation?
From Biochemistry and Molecular Biology of Plants
(W.Gruissem, B. Buchanan and R.Jones p.416. [/425
ASPP, Rockville MD, 2000
Slide 3.5
˚The DNA of most organisms encodes between ~5000 and
~500,000 proteins.
˚A rough definition of a gene is a stretch of DNA that encodes
one protein (polypeptide).
•To allow different cell type to form, or for an organism to
respond to changing conditions, only a subset of genes can be
“expressed” (actively organizing amino acid chain formation)
in any one cell or time. Therefore, genes must be switched
“on” and “off”.
•There are many types of controls on gene/protein expression.
What is the most common mechanism to turn on/off the
activity of a gene?
-to allow tRNA to recognize the genetic code, the
instruction template must be single-stranded
-the instruction template is not double-stranded DNA
but it is single-stranded mRNA
-it is the production of mRNA (called “transcription”)
which is the most common on/off level of gene control
The default state is “off”. Why?
From Intro to Genetic Analysis, 6th ed., p.499
A. Griffiths et al. W.H. Freeman and Co Pubs
.
Slide 3.6
The default gene state is “off”. Why?
Slide 37
From Intro to Genetic Analysis, 6th ed., p.499
A. Griffiths et al. W.H. Freeman and Co Pubs
.
•genome = sum of all the DNA of an organism demo
•rice= ~450 million base pairs
•average rice gene ~ 2000 bp
•corn = ~2.6 billion base pairs (humans = 2.9 billion bp)
•1cm of DNA stretched = 3 million bp
•rice genome stretched = 1.5m; corn = 8.7m
•plant cell nucleus L: 5-20 µm (=1/200-1/50 mm L)
•To accommodate DNA inside a nucleus, it is highly packaged as
“chromatin” (DNA wrapped around protein)
Therefore, for a gene to be functional, the DNA must first be
actively unpackaged and then the DNA strands must be unzipped.
Then an enzyme must read the new single-stranded DNA
template and synthesize mRNA. This entire process is called
transcription.
The unpackaging/unzipping factors are called transcription
factors. The enzyme which synthesizes mRNA=RNA polymerase.
Transcription factor -binding + activating protein
-recruits RNA Polymerase II
-recruits enzymes to unwind DNA
RNA Polymerase -enzyme for RNA transcription
-multiple polymerases at once
Transcriptisome= 20-30 protein complex needed
to start transcription:
Transcriptisome
complex
From Biochemistry and Molecular Biology of Plants
(W.Gruissem, B. Buchanan and R.Jones p.341, 343
ASPP, Rockville MD, 2000
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The TFs themselves must be synthesized and activated,
often by extracellular signals which alter their protein
conformation to make them functional. (topic of next lecture).
The transcription factors and RNA polymerase bind DNA by
recognizing specific double-stranded sequences, eg. TAATA
is the binding site for certain RNA polymerases.
Slide 3.8
How Transcription Factors Bind DNA
Specific amino acids interact with specific nucleotides of DNA,
because each nucleotide has its own specific shape/charges.
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From Introduction to Protein Structure p.145
C. Branden and J. Tooze
Garland Publishing, New York, 1999
DNA
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Protein
From Introduction to Protein Structure p.198
C. Branden and J. Tooze
Garland Publishing, New York, 1999
Slide 3.9
Transcription factors typically bind to DNA at specific sequences
in a region known generally as the “regulatory region”
(“promoter”, “enhancer”) located near the “transcribed region”.
Regulatory
Transcribed Region
Region
Gene=coding/transcribed region and regulatory region
Plant promoter = <500 bp, immed. Upstream = “gene switch”
Enhancer - DNA, location independent (+100kb)
Terminator - signal for transcription termination
+1 - start of transcription
•Remarkably, regulatory regions are modular - they can
be swapped between different genes. What are the
consequences of this “portability” for evolution?
•The transcribed region is interrupted by introns, ancient DNA
parasites which must be spliced out. The intervening true
Coding regions are called exons.
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From Biochemistry and Molecular Biology of Plants
(W.Gruissem, B. Buchanan and R.Jones p.324
ASPP, Rockville MD, 2000
Gene (definition): A regulated region of DNA consisting of
co-transcribed exons.
Slide 3.10
Post-Transcriptional RNA Intron Splicing
•RNA splicing of introns = lost
•Spliceosome= enzyme complex that recognizes the
intron/exon boundaries of mRNA and removes introns
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From Biochemistry and Molecular Biology of Plants
(W.Gruissem, B. Buchanan and R.Jones p.302
ASPP, Rockville MD, 2000
How are introns useful for evolution?
•Alternative splicing =new exon combinations
•After splicing, the mature spliced transcript is
exported from the nucleus to the cytoplasm for
translation into a protein.
Slide 3.11
The Eukaryotic Gene
From Biochemistry and Molecular Biology of Plants
(W.Gruissem, B. Buchanan and R.Jones p.340
ASPP, Rockville MD, 2000
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The Central “Dogma” of
Molecular Genetics
From Intro. To Genetic Analysis (ed 6) Griffiths et al
WH Freeman and Co. Publishers, NY 1996
Slide 3.12
Transcript ion, Translat ion and Signalling
Signal
source: M. Raiz ada
Kinase
Intracellular
Change in
Receptor
Conformation
Inactive
MembraneAnchored
Receptor
ATP
ADP P
Diffusible
Secondary
Messenger
ADP
ATP
Kinase
CYTOPLASM
Kinase
Highly
Packaged
Chromosome
P
PP
Kinase
NUCLEUS
Activated
TF
Bound
to DNA
Inactive
Transcription
Factors
Kinase
RNA
Pol ymerase
Kinase
P
Signal
Kinase Amplification
in Signal
Kinase
Transduction
Cascade
Ca+
Targetting of
protein to correct
compartment
or secretedl
-
E
I
E
I
E
I
E
I Terminator
DNA Coding R egion
Single-stranded
messenger
RNA
Splcing
Enzymes
(Splicesome)
Introns
spliced
out and lost
+
codon 1 2 ....
ACGUAG...
ACGUAG...
UGC AUC
aa1
t ransf er RNA AUC
at t ached t o
amino acid
aa2
+
Chaperone
allows correct
3-D folding
of protein
to create
enzyme
active site,
etc.. Allows
amino acid
interactions
protected
against
aqueous
environment
-
Mature
(spliced)
mRNA
UGC
+
Unwound 2-strand DNA
E I
ant icodon
Post-translational
modifications
(eg. Phosphorylation
or Calcium)
activate/repress
protein by altering
conformation.
P
RNA
Pol ymerase
Enhancer Promoter Exon Intron Exon I E I
Regulatory
DNA Region
Kinase
PP
Kinase
PP
PP
Ribosome
aa1
aa2
Unfolded
amino chain
(20 possible
amino acids)
Ribosome enz ymes
hold t RNA and mRNA
t o ext end amino acid chain.
Amino acids joined v ia
poly pept ide bonds.
Slide 3.13
To summarize, what are the key steps involved in
expressing a protein?
1. Transcription factors bind to DNA at regulatory
regions, unpackage and unzip.
2. Transcribe mRNA in nucleus (eukaryotes)
3. If introns, then remove by spliceosome.
4. Export mRNA into cytoplasm (for eukaryotes)
5. tRNA-mRNA codon binding in Ribosome complex
In cytoplasm.
6. Proteins must fold -- aided by another protein
complex, the “chaperone”.
7. Proteins localize to the correct compartment or are
secreted.
Slide 3.14
Post-Translation
•Post-translational modifications - modify surface
charges (sugars, phosphates, etc)
-covalent bonds, 100 types!!
•Correct 3-D protein folding: demo
-to create correct enzyme active site and shape
-only <1000 folds in all of life!!!
-DNA is rigid, but amino acid peptide bonds
can rotate, so many combinations
-other protein complexes (chaperones) assist in
folding in a destablizing aqueous environment
--chaperone
From Biochemistry and Molecular Biology of Plants
(W.Gruissem, B. Buchanan and R.Jones p.438
Slide 3.15
Protein enzymes can adopt multiple shapes
TIM Barrel - Rubisco
Horsheshow - RNasin
Beta roll - transcription factor
Beta barrell - GFP
Slide 3.16
After they fold, proteins must localize to the correct
compartment
•amino acid signal targetting sequence to correct
compartment
•at each membrane, recognition proteins bind aa signal
Compartments
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From Biochemistry and Molecular Biology of Plants
(W.Gruissem, B. Buchanan and R.Jones p.3, p.442
Slide 3.17
A Regulatory Module
•all cell types in an organism have same DNA/genes
•specialization of cell types by different sets of genes
turning on and off demo
•environmental responses (pathogens,drought) may use
different sets of genes; any trait requires many genes
•each set = module
•each module uses a specific set of transcription factors
binding to specific enhancer sequences at different
genes
Chromosome
ON
in leaf cell
OFF
Chromosome
in root cell
New molecular methods exist (gene chip microarray)
to discover the on/off status of thousands
of genes at once to define a transcriptional module
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From Intro to Genetic Analysis, 6th ed., Fig.14.26 A. Griffiths et al. W.H. Freeman and Co Pubs
.
Slide 3.18
Structure of an Arabidopsis Plant Gene/Genome:
Human data: IHGSC (2000) Nature 409, 860-921
Arabidopsis data: TAGI (2000) Nature 408, 796-814
•Genome size - 124 million bases (corn = 2.6 billion)
•#Genes - ~30,000
• Average gene density = 1 gene/4000 bp
Average gene (Ex+In)= 2000 bp (2kb) (humans=27kb)
•Average #exons/gene = 5 (humans = 8.8)
•Avg exon = 250 bp (humans = 145 bp)
•Average #introns/gene = 4
•Avg intron =150-180 bp (humans 3365bp; 60-30,000)
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From Biochemistry and Molecular Biology of Plants
(W.Gruissem, B. Buchanan and R.Jones p.324
ASPP, Rockville MD, 2000
Slide 3.19
Why did evolution select DNA to form mRNA to
form proteins? Why not simply use DNA to make
proteins directly after unzipping?
-RNA was the first molecule in evolution
-gives more control
-but theoretically DNA to protein directly is possible
-------------------------------------------------------------Concepts/Themes from Lecture 3
Many places of control:
•Why was DNA chosen for the genetic code of life?
•Transcription, splicing, mRNA export, translation,
post-translational modifications, protein folding,
compartment export/import
•DNA -- mRNA--- protein. Why??
•life is an orchestra of gene regulation as modules
(on/off/volume switches)
•in evolution, slight changes in the regulatory
switches have been used to create the diversity
found in life and in agriculture
Slide 3.20