DNA & RNA- Nucleic Acids and Protein Synthesis

IB BiologyCh. 16: Campbell

Ch. 5&6: Orange Book

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.

Introduction• Your genetic endowment is the DNA you inherited

from your parents.• Nucleic acids are unique in their ability to direct their

own replication.• The resemblance of offspring to their parents

depends on the precise replication of DNA and its transmission from one generation to the next.

• Once T.H. Morgan’s group showed that units of heredity are located on chromosomes, the two constituents of chromosomes - proteins and DNA - were the candidates for the genetic material.

• Until the 1940s, the great heterogeneity and specificity of function of proteins seemed to indicate that proteins were the genetic material.

• However, this was not consistent with experiments with microorganisms, like bacteria and viruses.

Discovery of DNA• 1868: Miescher first isolated

deoxyribonucleic acid, or DNA, from cell nuclei

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 the injected DNA of the phage provides the genetic information

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

So, for DNA, the amt. of A = T, andThe amt. of C= G (Chargaff’s Rules)

Who Discovered the Shape of DNA?

• James Watson and Francis Crick (1954) are credited with finally piecing together all the information previously gathered on the molecule of DNA. 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.

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.

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• One strand runs 5’ to 3’ while the other

runs 3’ to 5’ • Nucleotides are added on the 3’ end of

the original strand.• The new DNA strand forms and grows in

the 5’ 3’ direction only

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 growing 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

• The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells

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.• **Ligase- Connects the Okazaki fragments.

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