agr2451 lecture 3 (m. raizada) questionnaire -useful this week’s reading on reserve: plants, genes...

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 reactivemolecules 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 changein the local charge or size can cause changes in proteinconformation or binding.

4. The addition or loss of small molecules (phosphates,lipids, glucose) can be used as an “on/off” switch forprotein 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

From Chapter 11 Intro to Genetic Analysis

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 acidsto 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.

Slide 3.2

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.

From Chapter 11 Intro to Genetic Analysis

Chemical Features of DNA

Slide 3.3

Inheritance of the Instruction Book of Life (DNA)

Slide 3.4

•Need 1 DNA strand for instructions, but 2 strands forinheritance. Why?•Because of H-bonding, each strand can act as amirror-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-Chydrogen bonding as the set of instructions

P.322 Griffiths

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Introduction to Genetic Analysis (6th ed)Chapter 11 A.J. Griffiths et al.WH Freeman and Co. Publishers, NY, 1996

One strand of nucleic acid (mRNA) is the organizer of protein formation

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From Biochemistry and Molecular Biology of Plants(W.Gruissem, B. Buchanan and R.Jones p.416. [/425

ASPP, Rockville MD, 2000

•codon - 3 bp RNA sequence that encodesan amino acid•basis of genetic code

•tRNA binds an amino acid and carries it to the mRNA template, whereH-bonding between 3 letters of tRNA and mRNA allow correct placement of amino acid in the growingamino acid chain

•3 positions needed for code, because 20 different amino acids. A two Position code only has 16 combo’sgiven 4 possible letters.

•the amino acid polymer is built inside the Ribosome complex consisting ofmRNAs, tRNAs,

•”Translation” - conversionof 4 base genetic code to 20 amino acid protein code

•What was the key invention byevolution to allow DNA/RNA toorganize protein formation?

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 DNAbut it is single-stranded mRNA-it is the production of mRNA (called “transcription”) which is the most common on/off level of gene controlThe 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?

•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 calledtranscription.

The unpackaging/unzipping factors are called transcription factors. The enzyme which synthesizes mRNA=RNA polymerase.

From Intro to Genetic Analysis, 6th ed., p.499

A. Griffiths et al. W.H. Freeman and Co Pubs.

Slide 37

Transcription factor -binding + activating protein -recruits RNA Polymerase II -recruits enzymes to unwind DNA



From Biochemistry and Molecular Biology of Plants(W.Gruissem, B. Buchanan and R.Jones p.341, 343


Transcriptisomecomplex

RNA Polymerase -enzyme for RNA transcription -multiple polymerases at once

Transcriptisome= 20-30 protein complex needed to start transcription:

The TFs themselves must be synthesized and activated,often by extracellular signals which alter their proteinconformation to make them functional. (topic of next lecture).

The transcription factors and RNA polymerase bind DNA byrecognizing specific double-stranded sequences, eg. TAATAis the binding site for certain RNA polymerases.

Slide 3.8

Specific amino acids interact with specific nucleotides of DNA, because each nucleotide has its own specific shape/charges.

How Transcription Factors Bind DNA





Protein

DNA

From Introduction to Protein Structure p.145C. Branden and J. ToozeGarland Publishing, New York, 1999

From Introduction to Protein Structure p.198C. Branden and J. ToozeGarland Publishing, New York, 1999 Slide 3.9

Gene=coding/transcribed region and regulatory regionPlant 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 canbe swapped between different genes. What are theconsequences of this “portability” for evolution?•The transcribed region is interrupted by introns, ancient DNAparasites which must be spliced out. The intervening trueCoding regions are called exons.

Regulatory Region

Transcribed Region

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”.



From Biochemistry and Molecular Biology of Plants(W.Gruissem, B. Buchanan and R.Jones p.324


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

•Alternative splicing =new exon combinations

•After splicing, the mature spliced transcript is exported from the nucleus to the cytoplasm for translation into a protein.







How are introns useful for evolution?

Slide 3.11



The Eukaryotic Gene From Biochemistry and Molecular Biology of Plants(W.Gruissem, B. Buchanan and R.Jones p.340


The Central “Dogma” of Molecular GeneticsFrom Intro. To Genetic Analysis (ed 6) Griffiths et al. WH Freeman and Co. Publishers, NY 1996

Slide 3.12

ATP

ADP

Kinase

P

Signal

InactiveMembrane-AnchoredReceptor

IntracellularChange inReceptorConformation

Kinase

P

DiffusibleSecondaryMessenger

Kinase

Kinase

Kinase

Kinase

PP

SignalAmplificationin SignalTransductionCascade

Kinase

P

ATPADP

Kinase

PP

HighlyPackagedChromosome

KinasePP

RNAPoly-merase

InactiveTranscriptionFactors

+-

ActivatedTFBoundto DNA

KinasePP

RNAPoly-merase

Single-strandedmessengerRNA

SplcingEnzymes(Splicesome)

Mature(spliced)mRNA

Unwound 2-strand DNA

RegulatoryDNA Region

DNA Coding RegionEnhancer Promoter Exon Intron Exon I E I E I Terminator

ACGUAG...

aa2

AUC

aa1

UGC

ACGUAG...Ribosome enzymeshold tRNA and mRNAto extend amino acid chain.Amino acids joined viapolypeptide bonds.

aa1aa2

UGC AUC

codon 1 2....

anticodon

transfer RNAattached toamino acid

+

-

Unfoldedamino chain(20 possibleamino acids)

Chaperoneallows correct3-D foldingof proteinto createenzymeactive site,etc.. Allowsamino acidinteractionsprotectedagainstaqueousenvironment

+-P

Post-translationalmodifications(eg. Phosphorylationor Calcium)activate/repressprotein by alteringconformation.

Targetting ofprotein to correctcompartmentor secretedl

Intronssplicedout and lost

Transcription, Translation and Signallingsource: M. Raizada

Ca+

Ribosome

E I E I E I E I

NUCLEUS

CYTOPLASM

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 complexIn cytoplasm.

6. Proteins must fold -- aided by another protein complex, the “chaperone”.7. Proteins localize to the correct compartment or aresecreted.

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

Slide 3.15

Protein enzymes can adopt multiple shapes

Horsheshow - RNasinTIM Barrel - Rubisco

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

From Biochemistry and Molecular Biology of Plants(W.Gruissem, B. Buchanan and R.Jones p.3, p.442



Compartments

Slide 3.17

•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 usedifferent 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

A Regulatory Module

ON

OFF

Chromosomein leaf cell

Chromosomein root cell



New molecular methods exist (gene chip microarray)to discover the on/off status of thousandsof genes at once to define a transcriptional module

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:

•Genome size - 124 million bases (corn = 2.6 billion)•#Genes - ~30,000• Average gene density = 1 gene/4000 bpAverage 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)

Human data: IHGSC (2000) Nature 409, 860-921Arabidopsis data: TAGI (2000) Nature 408, 796-814





Slide 3.19

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

Why did evolution select DNA to form mRNA toform proteins? Why not simply use DNA to makeproteins directly after unzipping?-RNA was the first molecule in evolution-gives more control-but theoretically DNA to protein directly is possible--------------------------------------------------------------

Slide 3.20

agr2451 lecture 3 (m. raizada) questionnaire -useful this week’s reading on reserve: plants, genes...

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