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DNA & RNA- Nucleic Acids and Protein Synthesis
IB BiologyCh. 16: Campbell
Objectives
• Describe the history behind the discovery of DNA and its function
• Outline the structure of a nucleotide• Describe the structure of the DNA
molecule• Describe the process of DNA replication
including the various enzymes and that it is a semi-conservative process.
Discovery of DNA• In 1868, Friedrich Meischer first isolated
deoxyribonucleic acid from cells in pus and from fish sperm.
• No one knew DNA’s function.
Fredrick Griffith- 1928• First suggestion that about what genes are made of. • Worked with: 1) Two strains of Pneumococcus bacteria:
Smooth strain (S) Virulent (harmful) Rough strain (R) Non-Virulent
2) Mice-were injected with these strains of bacteria and watched to see if the survived.
3) Four separate experiments were done:-injected with rough strain (Lived)-injected with smooth strain (Died)-injected with smooth strain that was heat killed (Lived)-injected with rough strain & heat killed smooth (????)
Living S cells (control)
Living R cells (control)
Heat-killed S cells (control)
Mixture of heat-killed S cells and living R cells
Mouse diesMouse dies Mouse healthy Mouse healthy
Living S cells
RESULTS
EXPERIMENT
Griffith’s Conclusion• Somehow the heat killed smooth bacteria
changed the rough cells to a virulent form.• These genetically converted strains were called
“Transformations”• Something (a chemical) must have been
transferred from the dead bacteria to the living cells which caused the transformation
• Griffith called this chemical a “Transformation Principle”
Next Breakthrough came from the use of Viruses
• Viruses provided some of the earliest evidence that genes are made of DNA
• Molecular biology studies how DNA serves as the molecular basis of heredity
• Are only composed of DNA and a protein shell.
T2 Bacteriophage- a typical virus
Phage reproductive cycle
Figure 10.1C
Phage attaches to bacterial cell.
Phage injects DNA.
Phage DNA directs host cell to make more phage DNA and protein parts. New phages assemble.
Cell lyses and releases new phages.
Photo of T2 VirusesFig. 16-3
Bacterial cell
Phage head
Tail sheath
Tail fiber
DNA
100
nm
Hershey-Chase Experiment- 1952
• In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2
• To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection
• **They concluded that DNA provides the genetic information of a virus.
Fig. 16-4-1
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive protein
Radioactive DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Fig. 16-4-2
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive protein
Radioactive DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Empty protein shell
Phage DNA
Fig. 16-4-3
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive protein
Radioactive DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Empty protein shell
Phage DNA
Centrifuge
Centrifuge
Pellet
Pellet (bacterial cells and contents)
Radioactivity (phage protein) in liquid
Radioactivity (phage DNA) in pellet
Video Clip of Hershey-Chase• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#
Erwin Chargaff- 1950
Already known- DNA is a polymer of nucleotides- nitrogen base, pentose sugar, and a phosphate group.
Chargaff noticed a ratio of the bases:30.3% Adenine30.3% Thymine19.5% Guanine19.9% Cytosine
Chargaff’s Rules:The amount of A = T, and the amount of C = G.
Who Discovered the Shape of DNA?
• James Watson and Francis Crick (1954). They established the structure as a double helix - like a ladder that is twisted. The two sides of the ladder are held together by hydrogen bonds.
How did they get to their conclusions?
• They built many models, always perplexed at how it fit together, until one day, when they wandered into the office of a fellow scientist, Dr. Rosalind Franklin.
• She concluded: – DNA exists as a
long, thin molecule of uniform diameter
– The structure is highly repetitive
– DNA is helical
Rosalind Franklin used x-ray diffraction techniques to produce images of DNA
moleculesX-ray Diffraction
Along with Dr. Maurice Wilkins, she had taken x-ray crystallography photos of DNA. They saw her photos and realized the great secret- that DNA was coiled like a spring. They then made their model and won the Nobel Prize in 1962.
Franklin’s famous photo of DNA
So, What is DNA?Deoxyribonucleic Acid
• blueprint of life (has the instructions for making an organism)
• codes for your genes • made of repeating subunits called
nucleotides • shape is the double helix (twisted ladder)
I. Structure of DNA- 3 parts:
1. Sugar- Deoxyribose2. Phosphate Group3. Nitrogen bases
The sugar and phosphates make up the "backbone" of the DNA molecule.
Nucleotide
DNA is a nucleic acid, made of long chains of nucleotides- Sugar, phosphate, nitrogen base.
DNA and RNA are polymers of Nucleotides
Figure 10.2A
Nucleotide
Phosphate group
Nitrogenous base
Sugar
Polynucleotide Sugar-phosphate backbone DNA nucleotide**
Phosphategroup
Nitrogenous base(A, G, C, or T)
Thymine (T)
Sugar(deoxyribose)
Nitrogen basesBases come in two types: a. Purines (adenine and guanine- A&G) b. Pyrimidines (thymine and cytosine-
T&C).
DNA Maintains a Uniform Diameter• See pg. 310
• Watson and Crick reasoned that the pairing was more specific, dictated by the base structures
• They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C)
• The Watson-Crick model explains Chargaff’s rules: Adenine pairs to Thymine (A-T)Guanine pairs to Cytosine (G-C)**Very important- remember this!!!!
Base Pairing- Chargaff’s Rules
The two sides of the helix are held together by Hydrogen bonds (weak)
The sides of the DNA, the sugar and phosphate, are held together with covalent bonds (strong).
DNA Bonding
Simple Diagram of DNA
-Think of it like a
ladder, the bases being the rungs.
• Each strand of the double helix is oriented in the opposite direction
• The ends are referred to as the 3’ and 5’ ends.
Figure 10.5B
5 end 3 end
3 end 5 end
P
P
P
PP
P
P
P
Summary:• Chargaff
ratio of nucleotide bases (A=T; C=G)
• Watson & Crick (Wilkins, Franklin)
• The Double Helix √ nucleotides: nitrogenous base (thymine, adenine, cytosine, guanine); sugar deoxyribose; phosphate group
Putting it all together: here are some images of what the DNA double helix looks like:
DNA Replication and Repair
• The relationship between structure and function is manifest in the double helix
• Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material
The Basic Principle: Base Pairing to a Template Strand
• Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication
• In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules
Fig. 16-9-1
A T
GC
T A
TA
G C
(a) Parent molecule
Fig. 16-9-2
A T
GC
T A
TA
G C
A T
GC
T A
TAG C
(a) Parent molecule (b) Separation of strands
Fig. 16-9-3
A T
GC
T A
TA
G C
(a) Parent molecule
A T
GC
T A
TAG C
(c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand
(b) Separation of strands
A T
GC
T A
TA
G C
A T
GC
T A
TAG C
Three Proposed Models of DNA Replication
• Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand
• Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new)
DNA Replication: A Closer Look
• The copying of DNA is remarkable in its speed and accuracy
• More than a dozen enzymes and other proteins participate in DNA replication
DNA replication depends on specific base pairing
• In DNA replication, the strands separate– Enzymes use each strand as a template to
assemble the new strands
Parental moleculeof DNA
Both parental strands serveas templates
Two identical daughtermolecules of DNA
Nucleosomes
Anti-parallel Structure of DNA
Antiparallel nature• 5’ end corresponds to the Phosphate end• 3’ end corresponds to the –OH sugar • Replication runs in BOTH directions• In the original, one strand runs 5’ to 3’
while the other runs 3’ to 5’ • ** The new DNA strand forms and grows
in the 5’ 3’ direction only• So, nucleotides are added on the 3’ end of
the original strand.
5’ end
3’ end5’ end
Building New Strands of DNA
Building New Strands of DNA
• Each nucleotide is a triphosphate:(GTP, TTP, CTP, and ATP)
• Nucleotides only add to the 3’ end of the original strand (never on the 5’ end)
• Two phosphates are released (exergonic) and the energy released drives the polymerization process.
Getting Started• Replication begins at special sites called
origins of replication, where the two DNA strands are separated, opening up a replication “bubble”
• A eukaryotic chromosome may have hundreds or even thousands of origins of replication
• Replication proceeds in both directions from each origin, until the entire molecule is copied
Fig. 16-12b
0.25 µm
Origin of replication Double-stranded DNA molecule
Parental (template) strandDaughter (new) strand
Bubble Replication fork
Two daughter DNA molecules
(b) Origins of replication in eukaryotes
Fig. 16-12Origin of replication Parental (template) strand
Daughter (new) strand
Replication forkReplication bubble
Two daughter DNA molecules
(a) Origins of replication in E. coli
Origin of replication Double-stranded DNA molecule
Parental (template) strandDaughter (new) strand
Bubble Replication fork
Two daughter DNA molecules
(b) Origins of replication in eukaryotes
0.5 µm
0.25 µm
Double-strandedDNA molecule
Getting Started- Enzymes• At the end of each replication bubble is a
replication fork, a Y-shaped region where new DNA strands are elongating
• Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands
• Helicases are enzymes that untwist the double helix at the replication forks
• Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template
Fig. 16-13
Topoisomerase
Helicase
PrimaseSingle-strand binding proteins
RNA primer
55
5 3
3
3
• DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end
• The initial nucleotide strand is a short RNA primer
RNA Primers
• An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template
• The primer is short (5–10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand.
Synthesizing a New DNA Strand
• Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork
• Most DNA polymerases require a primer and a DNA template strand
How DNA daughter strands are synthesized
5 end
P
P
Parental DNA
Figure 10.5C
DNA polymerasemolecule
53
35
35
Daughter strandsynthesizedcontinuously
Daughter strandsynthesizedin pieces
DNA ligase
Overall direction of replication
53
• The daughter strands are identical to the parent molecule
Laying Down RNA Primers
III
I
DNA Replication-New strand Development
• Leading strand: synthesis is toward the replication fork (only in a 5’ to 3’ direction from the 3’ to 5’ master strand)
-Continuous• Lagging strand: synthesis is away from the replication fork
-Only short pieces are made called “Okazaki fragments”
- Okazaki fragments are 100 to 2000 nucleotides long-Each piece requires a separate RNA primer
-DNA ligase joins the small segments together (must wait for 3’ end to open; again in a 5’ to 3’ direction)
View video clip: • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#
DNA Replication Fork
Fig. 16-16a
OverviewOrigin of replication
Leading strand
Leading strand
Lagging strand
Lagging strand
Overall directions of replication
12
Key Enzymes Required for DNA Replication (pg. 314)
• Helicase - catalyzes the untwisting of the DNA at the replication fork
• SSBP’s - single stranded binding proteins, prevents the double helix from reforming
• Topoisomerase – Breaks the DNA strands and prevents excessive coiling
• Primase – synthesizes the RNA primers and starts the replication first by laying down a few nucleotides initially.
• **DNA Polymerase III - catalyzes the elongation of new DNA and adds new nucleotides on the 3’ end of the growing strand.
• **DNA polymerase I- Replaces the RNA primers with DNA.• **Ligase- Connects the Okazaki fragments.
Links to DNA Replication Animations
• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#
• http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html
Prokaryotic vs Eukaryotic Replication
• Prokaryotes– Have one single, circular loop of DNA (no free ends)– Contains 4 x 106 base pairs (1.35 mm)– (e coli has 4.6 million base pairs)– Rate for replication: 500 nucleotides per second– Only one origination point
Eukaryotic Replication• Eukaryotes w/Chromosomes:
-Have free ends-Humans have approx. 3 billion base pairs = 1 meter
-Rate for replication: 50 per second (humans)-Lagging strand is not completely replicated-Small pieces of DNA are lost with every cell cycle-End caps (Telomeres) protect and help to retain the genetic information-Each chromosome is one DNA molecule
Proofreading and Repairing DNA• Errors:
– Rate is one every 10 billion nucleotides copied– Proofreading is achieved by DNA polymerase (pg.
318)
• DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides
• In mismatch repair of DNA, repair enzymes correct errors in base pairing
• DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example)
• In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA
Proofreading and Repairing DNA1. Thymine dimer distorts the DNA
molecule
2. A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed.
3. Repair synthesis by a DNA polymerase fills in the missing nucleotides.
4. DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete.
DNA polymerase
DNA ligase
Nuclease
Telomeres• Short, non-coding pieces of DNA• Contains repeated sequences (ie. TTGGGG 20 times)• Can lengthen with an enzyme called Telomerase• Lengthening telomeres will allow more replications to occur.• Telomerase is found in cells that have an unlimited number of
cell cycles (commonly observed in cancer cells)• Artificially giving cells telomerase can induce cells to become
cancerous• Shortening of these telomeres may contribute to cell aging and
Apotosis (programmed cell death)
Ex. A 70 yr old person’s cells divide approx. 20-30X vs an infant which will divide 80-90X
Fig. 16-20
1 µm
Telomeres
A chromosome consists of a DNA molecule packed together with proteins
• The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein
• Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein
• In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid
Chromatin Packing In Eukaryotes• Chromatin is a complex of DNA and protein, and is
found in the nucleus of eukaryotic cells• Histones are proteins that are responsible for the first
level of DNA packing in chromatin• Nucleosomes- are like beads on a string, consist of DNA
wound twice around a protein core composed of two molecules each of the 4 main histone types.
Fig. 16-21a
DNA double helix (2 nm in diameter)
Nucleosome(10 nm in diameter)
Histones Histone tailH1
DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)
See p. 320
Fig. 16-21b
30-nm fiber
Chromatid (700 nm)
Loops Scaffold
300-nm fiber
Replicated chromosome (1,400 nm)
30-nm fiber Looped domains (300-nm fiber)
Metaphase chromosome
Part 2
RNA and Protein SynthesisCampbell: Ch. 17
I. Structure of RNA- 3 parts1. Sugar- Ribose2. Phosphate Group3. Nitrogen-containing bases
a. Adenineb. Uracil (substituted for Thymine)c. Guanined. CytosineAlso- RNA is single-stranded, unlike DNA.
- RNA is much smaller than DNA - RNA is in the nucleus and cytoplasm
A. 3 Types of RNA (used to build proteins)
1. Messenger RNA (mRNA)- carries the instructions from DNA to the ribosomes.
2. Transfer RNA (tRNA)- carries message from mRNA to find the specific amino acids.
3. Ribosomal RNA (rRNA)- makes up ribosomes, it puts the proteins together.
B. Transcription- The copying of information from DNA to RNA
Flow of Information is:DNA RNA Proteins
-Occurs in the nucleus.-RNA Polymerase is needed. -adds nucleotides to the 3’ end onlyThe base-pair rule is followed during
transcription, except, instead of pairing thymine with adenine, when creating an RNA strand, uracil is used
DNA Strand: 3’- T G C A T C A G A – 5’RNA Strand: 5’ -A C G U A G U C U – 3’
Only one strand of DNA (the template strand) is transcribed. (Antisense strand )The strand left un copied is the sense strandRNA nucleotides are available in the region of the chromatin (this process only occurs during Interphase)
Transcription begins on the area of DNA that contains the gene. Each gene has three regions:1. Promoter - turns the gene on or off2. Coding region - has the information on how to construct the protein3. Termination sequence - signals the end of the gene
RNA polymerase
RNA polymerase
Completed RNA
DNA of Gene
Initiation
Elongation
Termination
Promoter DNA Terminator
DNA
GrowingRNA
RNA Polymerase is responsible for reading the gene, and building the mRNA strand.**3 Steps: Initiation Elongation Termination
How it Works: Step One- Initiation
• RNA Polymerase binds to the “Promoter” region on the DNA (upstream about 25 nucleotides)
• RNA Polymerase recognizes this region because of the TATA box (sequence) on the antisense strand.
Elongation
• DNA is untwisted (hydrogen bonds are broken)
• About 10 base pairs are exposed• Nucleotides are are added to the 3’ end of
the growing mRNA molecule
• Proceeds at a rate of: 60 nucleotides/sec
Termination
• Termination site is reached by RNA Polyermase
• In Eukaryotes “AATAAA” is the signal• In Bacteria Translation can occur as it is
released from the first transcription event
• Final mRNA molecule is made consisting of “Coded” and “Non-coded” regions
Modification of pre mRNA
RNA is stable after a few modification steps.This mRNA is going into the cytoplasm
where there are many enzymes which would be detrimental to the messenger.
Or DON’T KILL THE MESSENGER!!!!!
So, Eukaryotic mRNA is processed before leaving the nucleus
• Noncoding segments called introns are spliced out
• A cap and a tail are added to the ends
DNA
RNAtranscriptwith capand tail
mRNA
Exon Intron IntronExon Exon
TranscriptionAddition of cap and tail
Introns removed
Exons spliced together
Coding sequence
NUCLEUS
CYTOPLASM
Tail
Cap
mRNA Structure• 1) 5’ cap: modified guanine; protection;
recognition site for ribosomes• 2) 3’ tail: poly(A) tail (adenine); protection;
recognition; transport• 3) RNA splicing: involves Introns & Exons• Exons (expressed sequences) retained• Introns (intervening sequences)
-These are spliced out / spliceosome, and the exons are kept.
Key Regions on Newly Transcribed mRNA
Transcription- The 3 Stages
DNA molecule
Gene 1
Gene 2
Gene 3
DNA strand
Polypeptide
Amino acid
Transcription
Translation
RNA
Codon
Protein Synthesis- Translation
• Occurs in the cytoplasm.• The genetic code is used to translate
mRNA into proteins. • Proteins are polymers, made of
polypeptides. • Each is made with a specific sequence of
amino acids
Translation Overview
Codons
• Each 3 bases of mRNA is called a codon, which translate to a single amino acid. (See codon chart).
• AUG is the start codon. This tells the ribosome to start making proteins.
Codon Chart- AUG is the start codon
Test for Understanding: A DNA sequence has the following bases: T A C - A G A - T T A - G G G - A T T What amino acids does it code for? (You'll need to use the codon chart)
rRNA or ribosomal RNARibosomes are the sites where the cell
assembles proteins according to genetic instructions
They consist of two parts, the large (50s) and small (30s) subunits, and are located either free floating in the cytoplasm or bound to the endoplasmic reticulum…
rRNA
tRNA• Transfer RNA (tRNA) is basically cloverleaf-shaped. • tRNA carries the proper amino acid to the ribosome when
the codons call for them. • At the top of the large loop are three bases, the anticodon,
which is the complement of the codon
Translation- Structure of a ribosome
rRNA- site of mRNA codon & tRNA anticodon couplingP site
holds the tRNA carrying the growing polypeptide chainA site
holds the tRNA carrying the next amino acid to be added to the chainE site
discharged tRNA’s
Ribosomes Build Polypeptides
Figure 10.12A-C
Codons
tRNAmolecules
mRNA
Growingpolypeptide
Largesubunit
Smallsubunit
mRNA
mRNAbindingsite
P site A site
P A
Growingpolypeptide
tRNA
Next amino acidto be added topolypeptide
Translation- Step 1- Initiation
• So here it goes:• In the first step in protein synthesis, the
small subunit of the ribosome binds to the mRNA molecule at the start codon
• The first tRNA delivers its amino acid• The larger unit of rRNA is also attached
2. Elongation• Begins when the next tRNA binds to the A
site of the ribosome • The first tRNA is released after the amino
acid is taken • The next tRNA moves from the A site to
the P site • and the used tRNA moves to the E site
where it is released • This process continues until it reaches the
stop codon.
3. Termination
• The ribosome reaches the stop codon.• The polypeptide is released along with
the two ribosomal units.
Figure 10.14
1 Codon recognition
Amino acid
Anticodon
AsiteP site
Polypeptide
2 Peptide bond formation
3 Translocation
Newpeptidebond
mRNAmovement
mRNA
Stopcodon
Figure 10.15 (continued)
4Stage ElongationGrowingpolypeptide
Codons
5Stage Termination
mRNA
Newpeptidebondforming
Stop Codon
The ribosome recognizes a stop codon. The poly-peptide is terminated and released.
A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time.
Polypeptide
And so,
• DNA is copied into mRNA inside the nucleus.
• The mRNA moves into the cytoplasm and tRNA and rRNA join up to read the message and produce a polypeptide chain
• This will be further processed into a protein
Translation Diagram
SummaryDNA Transcription RNA Translation Protein
DNA RNAOnly 1 type 3 typesDeoxyribose riboseA,C,G,T A,C,G,UIn nucleus in nucleus & cytoplasmMade by replication made by transcription-
mRNADNA codons RNA codons & anticodonsRelatively large Relatively smallDouble-Stranded Single-Stranded
Online animationsTranscription to Translation**http://207.207.4.198/pub/flash/26/transmenu_s.swf
Animated View of Transcriptionhttp://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html#
Protein Synthesis (simple)http://www.wisc-online.com/objects/index_tj.asp?objid=AP1302