Transcript CH 16-17: DNA, RNA & PROTEINS

DNA, 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
The Search for the Genetic Material: Scientific Inquiry
• 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 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 heatkilled remains of the pathogenic strain
– With living cells of the nonpathogenic strain,
some of these living cells became pathogenic
EXPERIMENT 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:
Living S
(control) cells
Living R
Heat-killed
(control) cells (control) S cells
Mixture of heat-killed S cells
and living R cells
RESULTS
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells
are found in
blood sample.
Figure 16.2
CONCLUSION Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an
unknown, heritable substance from the dead S cells.
The Search for the Genetic Material: Scientific Inquiry
• Griffith called the phenomenon transformation
– Now defined as a change in genotype and
phenotype due to the assimilation of external
DNA by a cell
Evidence That Viral DNA Can Program Cells
Additional evidence for DNA as the genetic material
came from studies of a virus that infects bacteria
Oswald Avery
• 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!
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
Phage
head
Tail
Tail fiber
Figure 16.3
Bacterial
cell
100 nm
DNA
• 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
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.
1 Mixed radioactively
labeled phages with
bacteria. The phages
infected the bacterial cells.
Phage
2 Agitated in a blender to 3 Centrifuged the mixture
separate phages outside
so that bacteria formed
the bacteria from the
a pellet at the bottom of
bacterial cells.
the test tube.
Radioactive Empty
protein
protein shell
Radioactivity
(phage protein)
in liquid
Bacterial cell
Batch 1: Phages were
grown with radioactive
sulfur (35S), which was
incorporated into phage
protein (pink).
Batch 2: Phages were
grown with radioactive
phosphorus (32P), which
was incorporated into
phage DNA (blue).
4 Measured the
radioactivity in
the pellet and
the liquid
DNA
Phage
DNA
Centrifuge
Radioactive
DNA
Pellet (bacterial
cells and contents)
Centrifuge
Radioactivity
(phage DNA)
Pellet
in pellet
RESULTS
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.
Figure 16.4
CONCLUSION
Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material.
• DNA structure and replication
• RNA
– Transcription
– Translation
• Protein synthesis
– Amino acids
DNA
• 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 phosphate,
• 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
A
C
G
T
A
G
G
T
T
Matching copy
T
G
C
A
T
C
C
A
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 are
assembled 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 Synthesis
Central Dogma of Molecular Biology
transcription
DNA
translation
RNA
Proteins
• Multiple forms of RNA molecules involved
in protein synthesis:
–
–
–
–
Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA
(tRNA)
Small Nuclear RNA (snRNA)
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
The Key to Making or Breaking a Heart
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
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.
Heart Maker
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.
SRF promotes cardiac muscle cell
development in mouse embryo. Blue
stain shows a developing heart in an
early mouse embryo. © 2007 Medical College of Wisconsin
SRF dimer (yellow & orange) bound to the SRE element of DNA (blue)
Transcription Factor Cooperation
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.
The Making of a Protein
Co-op
factor
Co-op factor
Co-op
factor
SRF
Heart Breaker
SRF
SRE
Cardiac
Muscle
Protein
DNA
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.
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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 Translation
1. 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
U
U
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 RAT
THE CAT SAW THE HAT
Frame Shift Mutation: Entire Sequence is disrupted
• Deletion: a small DNA segment is lost
THE CAT SAW THE RAT
THE ATS AWT HER AT
• Insertion: a segment of DNA is added
THE CAT SAW THE RAT
THE 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.