DNA, RNA & PROTEINSDNA, RNA & PROTEINS

The molecules of life

• Overview: Life’s Operating Instructions• In 1953, James Watson and Francis Crick (and others

acknowledged and not acknowledged) shook the world

– With an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA

Figure 16.1

• DNA, the substance of inheritance– Is the most celebrated molecule of our time– Most significant discovery in the history of science?

• Hereditary information– Is encoded in the chemical language of DNA and

reproduced in all the cells of your body

• It is the DNA program– That directs the development of many different types

of traits– Controls Cell Determination and Differentiation in

Living Organisms

• Concept 16.1: DNA is the genetic material

• Early in the 20th century– The identification of the molecules of inheritance

loomed as a major challenge to biologists

The Search for the Genetic Material: Scientific Inquiry

• The role of DNA in heredity– Was first worked out by studying bacteria and the

viruses that infect them

Evidence That DNA Can Transform Bacteria

• In 1928, Frederick Griffith was studying Streptococcus pneumoniae– A bacterium that causes pneumonia in mammals

• He worked with two strains of the bacterium– A pathogenic strain and a nonpathogenic strain

• Griffith found that when he mixed heat-killed remains of the pathogenic strain– With living cells of the nonpathogenic strain,

some of these living cells became pathogenic

Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below:

Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by anunknown, heritable substance from the dead S cells.

EXPERIMENT

RESULTS

CONCLUSION

Living S(control) cells

Living R(control) cells

Heat-killed(control) S cells

Mixture of heat-killed S cellsand living R cells

Mouse dies Mouse healthy Mouse healthy Mouse dies

Living S cellsare found inblood sample.

Figure 16.2

• Griffith called the phenomenon transformation– Now defined as a change in genotype and

phenotype due to the assimilation of external DNA by a cell

The Search for the Genetic Material: Scientific Inquiry

Evidence That Viral DNA Can Program Cells

Additional evidence for DNA as the genetic material came from studies of a virus that infects bacteria

• 1940’s-50’s. Oswald Avery conducts an experiment in Bacterial Transformation. Taking Streptococcus bacteria, he mixed live, nonvirulent ones with dead (virulent) bacteria.

• The result was that some of the live bacteria became virulent.

• Avery grew virulent bacteria, then broke open the cells. He attempted to isolate the “Transforming Principle” or active component. DNA!

Oswald Avery

Maurice Wilkins and Rosalind Franklin

• Were the first to obtain very good x-ray diffration images of the DNA fibers.

Erwin Chargaff • was a biochemist who first figured out the equation

for the different bases. Here is what he concluded:

• the amount of (A)denine will always equal the amount of (T)hymine and the amount of (G)uanine will always equal the amount of (C)ytosine.

• Viruses that infect bacteria, bacteriophages– Are widely used as tools by researchers in

molecular genetics

Figure 16.3

Phagehead

Tail

Tail fiber

DNA

Bacterialcell

100

nm

• Alfred Hershey and Martha Chase– Performed experiments showing that DNA is

the genetic material of a phage known as T2

• The Hershey and Chase experiment

In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells.

Radioactivity(phage protein)in liquid

Phage

Bacterial cell

Radioactiveprotein

Emptyprotein shell

PhageDNA

DNA

Centrifuge

Pellet (bacterialcells and contents)

RadioactiveDNA

Centrifuge

Pellet

Batch 1: Phages weregrown with radioactivesulfur (35S), which wasincorporated into phageprotein (pink).

Batch 2: Phages weregrown with radioactivephosphorus (32P), which was incorporated into phage DNA (blue).

1 2 3 4Agitated in a blender toseparate phages outsidethe bacteria from thebacterial cells.

Mixed radioactivelylabeled phages withbacteria. The phagesinfected the bacterial cells.

Centrifuged the mixtureso that bacteria formeda pellet at the bottom ofthe test tube.

Measured theradioactivity inthe pellet and the liquid

Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells. When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus.

Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material.

RESULTS

CONCLUSION

EXPERIMENT

Radioactivity(phage DNA)in pellet

Figure 16.4

• DNA structure and replication

• RNA– Transcription– Translation

• Protein synthesis– Amino acids

DNADNA• Deoxyribonucleic Acid (DNA) is the

blueprint for life: contains ALL the necessary information to make a new organism

DNA structure• DNA is a polymer of

nucleotides– Each nucleotide composed of

• a phosphatephosphate,

• a sugar (deoxyribose),

• and organic nitrogenous base.

Four DNA bases• Four kinds of nitrogenous bases:

Purine bases

A = Adenine

G = Guanine

Pyrimidine bases

T = Thymine

C = Cytosine

DNA: Complimentary base pairing• Adenine pairs with Thymine

A T

• Cytosine pairs with Guanine

C G

Each DNA strand is a compliment of the other

Original strand Matching copy A T

C GG CT AA TG CG CT AT A

DNA STRUCTURE

• DNA is a double helix

• Discovered by

Watson and Crick, 1953

DNA structure

Antiparallel Strands

DNA REPLICATION (in the nucleus)

• Each DNA strand becomes a template, parent strand becomes apart

• Proper base-pairs areassembled on that template(with proper enzymes:polymerase and ligase).

• There’s always a pool of nucleotides (A,C,T,G) in the nucleus

Semi-Conservative Replication

½ of the original is “conserved” and the other half is “new”

DNA replication• Nucleotides are connected together to make

a new strand that is complimentary to the old strand.

• The new double strand is

identical to the old double strand

• Semi-conservative replication:

half old, half new DNA

on each strand

RNA structure and synthesis• RNA: Ribonucleic Acid

• Is very similar to DNA

(repeating subunits, nucleotides).

• Difference between RNA and DNA:– Each nucleotide contains a different sugar

(ribose instead of deoxyribose)– Bases are A, G, C, and U (uracil, not thymine)

A pairs with U; G pairs with C

RNA

• RNA is single stranded

and shorter

• RNA is less stable than DNA:

RNA doesn’t persist in the cell for long (sometimes it exists for a few seconds),

whereas DNA can persist for the life of the cell.

Protein SynthesisCentral Dogma of Molecular Biology

DNARNA

Proteins• Multiple forms of RNA molecules involved

in protein synthesis:– Messenger RNA (mRNA)– Ribosomal RNA (rRNA)– Transfer RNA (tRNA)– Small Nuclear RNA (snRNA)

transcription

translation

Transcription: DNA RNA

• TRANSCRIPTION: RNA synthesis from DNA.

• Transcription: making an RNA copy, called messenger RNA (mRNA), of a small part of the DNA molecule.

Transcription• Transcription occurs in the Nucleus• mRNA carries the message about what type of

protein to make from the DNA in the nucleus to the ribosome

• The nucleotide sequences of RNA and DNA are the same (except in RNA uracil is used instead of thymine)

• mRNA is synthesized from DNA using base pairing

• DNA unwinds in a section

Transcription of RNA from a template strand of DNA

• RNA polymerase attaches at the promoter sequence of DNA, and it moves along the DNA, unzipping the strands – this allows for one mRNA molecule to be formed.

Transcription:

Promotors and Transcription Factors

Serum Response Factor (SRF) is a protein transcription factor. Transcription factors use the information on DNA to regulate RNA production that ultimately encodes for proteins the body needs. SRF promotes the formation and growth of cardiac muscle cells.

SRF functions as a "dimer" composed of two identical subunits. The SRF dimer works as a complex, in cooperation with other associated factors to help control gene expression. The number and type of SRF-associated factors determines which genes are expressed, where they are expressed, and when they are expressed. SRF and the other factors bind a DNA sequence known as the Serum Response Element (SRE). The SRE region is known for its characteristic nucleotide sequence and is found in the promoters of SRF responsive genes in many different species.

One way SRF is important for heart formation and function is based on its ability to regulate genes essential for the differentiation and growth of cardiac muscle cells. In mouse embryos, SRF is absolutely required for proper cardiac development. Research shows that embryos deprived of SRF die from underdeveloped hearts. Overexpression of SRF can result in cardiac hypertrophy (enlarged heart syndrome). Better understanding of SRF function holds the potential to develop therapies designed to repair human heart damage.

Serum Response Factor

One of SRF's main functions is to manage the expression of genes associated with cardiac muscle cells. Manipulating levels of SRF protein can have various effects on an organism. Research with developing mouse embryos showed that those deprived of SRF died early in development. In contrast, overexpression of SRF in adult mouse heart resulted in cardiac hypertrophy (enlarged heart syndrome) due to the excessive production of proteins involved in cardiac growth.

MUHS Smart Team: Wesley Borden, Daniel Brodzik, Patrick Carter, Brian Digiacinto, John Geary, Thomas Niswonger, Joseph Radke, Matthew Shields, and Caleb Vogt Teacher: Keith Klestinski; Mentors: Dr. Ravi Misra, PhD and Dr. Mary Holtz, PhD from the Medical College of Wisconsin Department of Biochemistry

The Key to Making or Breaking a Heart

Heart Maker

SRF promotes cardiac muscle cell development in mouse embryo. Blue stain shows a developing heart in an early mouse embryo.

SRF dimer (yellow & orange) bound to the SRE element of DNA (blue)

After SRF forms a dimer, it binds with different cooperating transcription factors. The whole complex binds to the SRE region of DNA and allows for transcription of mRNA. The mRNA made during transcription is then used to generate a specific protein.

Co-op factor

SRF SRF

SRE

DNA

Co-op factor

Co-op factor

Transcription Factor CooperationThe Making of a Protein

Copyright © 1994-2007 by Access Excellence @ the National Health Museum

Heart Breaker

http://maxshouse.com/Hypertrophic_%20Cardiomyopathy/Hypertrophic_Cardiomyopathy_Dia.jpg

© 2007 Medical College of Wisconsin 

mRNA Cardiac Muscle Protein

RNA Processing (Eukaryotes)• Once mRNA is formed, enzymes in the nucleus

remove the introns (non-coding regions) and leave the exons (expressed segments)

Splicosomes and snRNP’s

Resulting mRNA Strand:A product of RNA Processing

- G3P Cap- Poly A (Adenine) Tail- Polyadenylation

Signal- UTR’s (Un-translated

Regions)

Exons = Domains

Protein Synthesis – Transcription and RNA Processing

The Genetic Code• Each 3 consecutive bases on the mRNA is a code word,

codon, that specifies an amino acid.• The genetic code consists of 64 codons, (4x4x4), but

only 61 code amino acids. • Three codons act as

signal terminators

(UAA, UAG, UGA)

• One codon, AUG, codes

for methionine, and is also

the start signal for translation.

The Genetic Code• The Genetic Code – every three nucleotides on mRNA

codes for a particular amino acid (3 at a time)• Code is universal – true for all organisms!

20 Amino Acids

There are 20 amino

acids – they are like

the ‘bricks’ to make

all proteins

Translation• Translation: synthesizing a protein from amino

acids, according to the sequences of the nucleotides in mRNA.

• Occurs at the ribosomes, in cytoplasm of cell

• Ribosomal RNA, rRNA, is needed for protein synthesis – helps mRNA bind to ribosome.

• Transfer RNA, tRNA, brings specific amino acids to the ribosome to be assembled as proteins.

Translation• Ribosomal RNA, rRNA, joins with a number

of proteins to form ribosomes

• Ribosomes are the sites of protein synthesis

• Ribosomes consist of

a large subunit and

a small subunit.

mRNA binds to the

small subunit.

Transfer RNA (tRNA)• Transport molecule

that carries specific

amino acids to a

ribosome

(80 nucleotides long)

Folded

• Each tRNA recognizes

the correct codon on

the mRNA molecule

Steps in Translation

1. Initiation (begins with AUG start codon)

2. Elongation (addition of amino acids)

3. Termination (ends with stop codon – UAA UAG or UGA

Considerations in Translation1. mRNA leaves the nucleus and migrates to ribosome

2. mRNA binds to small ribosomal subunit

3. tRNA brings an amino acid to the ribosome, where anticodon on the tRNA binds to the codon of the mRNA

4. The amino acid bonds to its adjoining amino acid to form a growing polypeptide molecule

5. The tRNA without the amino acid is released from the ribosome

6. Other tRNA’s bring amino acids to the ribosome to complete the protein molecule

Ribosomal Structure

• Making proteins from RNA

UU

U

Bacterial (Prokaryotic):

Simultaneous Transcription/

Translation

Protein synthesis

• In cytoplasm of the cell

Protein synthesis

• Amino acids are the repeating sub-units of protein molecules.

• Amino acid order determines the protein

• 20 amino acids exist in all life forms

• Order of amino acids is important, determines the 3-dimensional shape of the molecule.

• Structure of the protein determines its function

Proteins• Biological activity (function) of proteins

depends largely on its 3-D structure

Genomic Geography

• In Cell Nucleus: RNA is produced by transcription.

• RNA is single-stranded; substitutes the sugar ribose for deoxyribose and the base uracil for thymine

• Messenger RNA or mRNA, conveys the DNA recipe for protein synthesis to the cell cytoplasm.

• mRNA binds to ribosome, each three-base codon of the mRNA links to a specific form of

transfer RNA (tRNA) containing the complementary three-base sequence.

• This tRNA, in turn, transfers a single amino acid to a growing protein chain.

• Each codon directs the addition of one amino acid to the protein. Note: the same amino acid can be added by different codons; in this illustration, the mRNA sequences GCA and GCC are both specifying the addition of the amino acid alanine (Ala).

Important• Both DNA and RNA have a direction: one

end is the 3’ the other is the 5’ end.• Thus, codons are read in one direction only.• DNA/RNA Polymerase read 3’ to 5’• M-RNA read 5’ to 3’ by the Ribosome• Also, note there is redundancy in the genetic

code: the different sequences can specify for the same amino acid.Example: UUA and UUG = Leucine

Gene Expression: From Start to Finish

When things go wrong…• Mutations: changes in the DNA sequence, that may be passed along to

future generations.

Point mutations:

• Substitution - a single base substitution

THE CAT SAW THE RATTHE CAT SAW THE HAT

Frame Shift Mutation: Entire Sequence is disrupted

• Deletion: a small DNA segment is lost

THE CAT SAW THE RATTHE ATS AWT HER AT

• Insertion: a segment of DNA is added

THE CAT SAW THE RATTHE BCA TSA WTH ERA T

Point Mutations: Missense vs. Nonsense

Frame Shift Mutations: Missense vs. Nonsense

Mutations• Frame-shift mutation: modification of the reading

frame after a deletion or insertion, resulting in all codons downstreams being different.

For example:THE RAT SAW THE CAT AND RAN

If you take out the “R” in “RAT” and shift the frames, you get:

THE ATS AWT HEC ATA NDR AN

The resulting sentence (or mRNA message) is meaningless!

Mutations

• Somatic mutations: occur in body cells, or cells that do not lead to gametes.

• Somatic mutations that occur in leaves, roots or stems are usually not passed on to future generations… UNLESS the plant is reproduced asexually.