Download Inquiry into Life Twelfth Edition

BL 426 Molecular Biology
• What is molecular biology?
– explain biological phenomena in molecular terms
– study gene structure, function at molecular level
• Melding of genetics, microbiology and biochemistry
• Dates from about 1940s to 1960s
• Techniques permitted incredible details of basic
science; many practical applications in medicine,
agriculture
1-1
Learning Outcomes for Students:
• Generally explain how science differs from other
ways of knowing – experimental basis for
conclusions
• Define major terms used in Molecular Biology
• Explain major organizing concepts in Molecular
biology.
• Recognize social and ethical relevance of content
covered in Molecular biology
• Analyze and present primary scientific data,
research by Nobel Laureates in Medicine or
Chemistry.
1-2
Chapt. 1
Brief History
1.1 Transmission Genetics (Mendelian):
• Transmission of traits from parents to offspring
• Chemical composition of genes discovered 1944;
Mendel didn’t know chromosomes
– Gene – particles contributed by parents
– Phenotype – observed characteristics
Mendel studied garden pea, detailed records,
statistics
Important Figure 3, Table 1
1-3
Mendel’s Laws of Inheritance
•
•
•
•
•
Genes exist in different forms - alleles
One allele (A) dominant over other, recessive (a)
Each parent carries 2 copies of gene: diploid for that gene:
Parents in 1st mating are homozygotes (AA, aa)
First filial generation (F1) contains offspring of parents
– Heterozygotes have one copy of each allele: (Aa)
• Sex cells, or gametes, are haploid, contain 1 copy of gene
• Heterozygotes produce gametes having either allele
• Homozygotes produce gametes having only one allele
– Recall Punnet square analysis to predict progeny
1-4
Chromosome Theory of Inheritance
• Chromosomes: discrete
physical entities that carry genes
• Morgan used fruit fly, Drosophila
melanogaster, to study genetics
• Autosomes occur in pairs in individual
• Sex chromosomes are X and Y
– Female has two X chromosomes
– Male has one X and one Y
1-5
Hypothetical Chromosomes
• Every gene has its place, or locus, on chromosome
centromere attaches to spindle
• Genotype: combination of alleles found in organism
• Phenotype: visible
expression of genotype
– Wild-type phenotype most common, generally
accepted standard
– Mutant alleles – altered,
usually recessive
Fig. 3
1-6
Genetic Recombination and Mapping
• Genes on separate chromosomes behave independently
• Genes on same chromosome behave as if linked
Genetic linkage is not absolute: permits mapping
• Recombination produces new combinations of alleles in
offspring, combinations not seen in parents
– from Crossing-over of chromosomes during meiosis
• Genetic Mapping: farther apart two genes are on
chromosome, more likely they are to recombine (Fig. 4)
• If 2 loci recombine with frequency of 1%: map distance is
1 centimorgan (named for Morgan)
• (mapping applies to Prokaryotes and Eukaryotes)
1-7
1.2 Molecular Genetics overview
• Discovery of DNA: general structure of nucleic acids
found by end of 19th century
Long polymers or chains of nucleotides
Nucleotides linked by sugars through phosphate groups
• Composition of Genes: In 1944, genes are
composed of nucleic acids
Genes perform three major roles:
• Replicate faithfully
• Direct production of RNAs and proteins
• Accumulate mutations, thereby allowing evolution
1-8
DNA Replication
• Franklin and Wilkins x-ray diffraction
data on DNA
• Watson and Crick proposed DNA is
double helix
– Two DNA strands wound around
each other
– Strands are complementary –
• if know sequence of one, automatically
know sequence of other
• Semiconservative replication:
one strand of parental double helix
conserved in each daughter double helix
1-9
Genes Direct Production of Polypeptides
• Defective gene gives defective or absent enzyme
• Early idea: one gene makes one enzyme
• Gene expression - process making gene product:
– Transcription: copy of DNA is made as RNA
– Translation: RNA copy is read or translated to
assemble a protein (on ribosomes)
– Codon: sequence of 3 nucleic acid bases that
stand for 1 amino acid
1-10
Genes Accumulate Mutations
Genes change in several ways:
•
•
•
•
Change one base to another
Deletions of one base up to a large segment
Insertions of one base up to a large segment
Rearrangements of chromosomes
• The more drastic changes make it more likely that
gene or genes involved will be totally inactivated
1-11
1.3 Three Domains of Life
Current research supports division of
living organisms into three domains
• Bacteria – typical prokaryotes:
E. coli; Thermus aquaticus
• Eukarya – nucleus, organelles:
yeast, amoeba, worms, mice, humans
• Archaea (prokaryotes) often live in
inhospitable regions of earth
• Thermophiles tolerate extremely
high temperatures Thermococcus
• Halophiles tolerate very high salt
concentrations Halobacterium
1-12
1-14
Chapt. 2 DNA is Genetic Material
Learning outcomes:
• Recall and explain basic experiments, concepts of
DNA as basis of heredity;
• Describe general structure of DNA and RNA:
• chain-like molecules composed of nucleotide subunits
• Nucleotides contain a base linked to the 1’-position of a
sugar and a phosphate group
• Phosphate joins sugars in DNA or RNA chain through 5’and 3’-hydroxyl groups by phosphodiester bonds
• (be able to draw Fig. 10 details)
Important Figures: 2, 4, 5, 6, 7, 8, 9, 10*, 11*, 13, 14*, 15, 20 Table 4
Review Q 2, 3, 4, 8; AQ 1, 2
1-15
DNA is Genetic Material
Bacterial Transformation – Griffith, 1928; Avery, 1944
Fig. 2
2-16
DNA Confirmation
• In 1940s geneticists doubted importance of DNA:
appeared monotonous repeats of 4 bases
• 1950 Chargaff showed 4 bases were not present in
equal proportions
• 1952 Hershey and Chase demonstrated (S35, P32)
that bacteriophage T2 infection comes from DNA
• 1953 Watson & Crick published double-helical
model of DNA structure
• Genes are made of nucleic acid, usually DNA
• Some simple genetic systems (viruses) have
RNA genes
2-17
DNA is phage T2 genetic material
Hershey – Chase 1952
Fig. 4
Phage T2
2-18
Purines and Pyrimidines
• A and G are purines; C, T and U are pyrimidines
• Note numbering of positions
Fig. 5
2-19
Nucleosides and Nucleotides
• RNA component parts
– Nitrogenous bases
• Uracil (U)
• replaces Thymine
– Phosphoric acid
– Ribose sugar
• Bases had ordinary numbers
• Carbons in sugars - primed numbers
Fig. 7
• Nucleosides lack phosphate
• Nucleotides contain phosphate
2-20
DNA nucleotide linkage
• Nucleotides are nucleosides with phosphate group
attached through phosphodiester bond
• Nucleotides may contain 1, 2, or 3 phosphate groups
Fig. 9
2-21
Trinucleotide:
phosphodiester bond
Polarity: 5’- T-C-A-3’
– Top of molecule has free
5’-phosphate group = 5’ end
– Bottom has free
3’-hydroxyl group = 3’ end
Figs. 10, 11
2-22
DNA Double Helix
• Twisted ladder structure:
– Curving sides of ladder are
sugar-phosphate backbone
– Ladder rungs are base pairs
– A-T and G-C hydrogen bond
– About 10 base pairs per turn
• Two strands are antiparallel
Fig. 13
2-23
Fig.
14
Genes can be made of RNA or DNA
Hershey & Chase investigated bacteriophage T2
(virus particle, DNA,package of genes)
– T2 has no metabolic activity of its own
– When virus infects host, cell makes viral proteins
– Viral genes are replicated, newly made genes with viral
coat proteins assemble into virus particles
Viruses are model systems for molecular biology:
• Some viruses contain DNA genes, either single- or doublestranded (M13, lambda)
• Some viruses have RNA genes, either single- or doublestranded (MS2, HIV, rabies)
2-24
DNA Content varies
among Organisms
• Ratios of G to C, A to T
are fixed in any organism
• But, total percentage of
G + C varies over a
range of 22 to 73%
• Differences in total G+C
reflected in differences in
physical properties
(such as melting temp)
2-25
Polynucleotide Chain
Hybridization
** Hybridization: process of
putting together combination
of two different nucleic acids
– Strands could be 1 DNA
and 1 RNA
– Could be 2 DNAs
– Could be complementary
or nearly complementary
sequences
– Valuable technique
Fig. 20
2-26
DNA Shapes and Sizes
DNA size is expressed 3 different ways:
– Number of base pairs (bp, kbp or kb)
– Molecular weight – 660 daltons (D) is average
molecular weight of 1 base pair
– Length – 33.2 Å per helical turn of 10.4 base pairs
DNA shape can be linear, circular (relaxed), or
covalently closed circular
Measure DNA size (shape) using electron microscopy
or gel electrophoresis
2-27
• Phage DNA is typically circular; so are bacterial chromosomes
• Some DNAs are linear – ex., eukaryotic chromosomes
2-28
• Supercoiled DNA coils or wraps around itself like a twisted rubber band
Ex. DNA Size and Genetic Capacity
Estimate how many genes are in a piece of prokaryotic DNA
• Gene encodes protein; avg. Protein is about 40,000 D (40 kD)
– How many amino acids does this represent?
• Average mass of an amino acid is about 110 D
• Average protein of 40,000 / 110 = 364 amino acids
• Each amino acid = 3 DNA base pairs
• 364 amino acids requires 1092 base pairs
E. coli chromosome = 4.6 x 106 bp; ~4200 proteins
– Phage l (infects E. coli) = 4.85 x 104 bp ~44 proteins
– Phage x174 (one of smallest) = 5375 bp ~5 proteins
2-29
Chapt. 3 Gene Function
Learning outcomes:
• Recall and explain basic processes in production
of polypeptide from DNA
–
–
–
–
–
–
transcription
translation
ribosome
tRNA
mRNA
polypeptide, protein structure (1o, 2o, 3o, 4o)
Important Figures: 1, 2a, 3, 4, 14, 16, 17, 18, 19, 20*, 26
1-30
Review Q: 1, 2, 4, 7, 9, 11, 12, 13*, 14*, 15*; AQ 1
Chapt. 3 Gene Function
3.1 Storing Information
Producing protein from DNA involves both
transcription and translation
– A codon is 3 base sequence
that determines what amino acid
– Template strand:
complementary DNA
strand used to
generate mRNA
– Nontemplate strand:
not used for RNA
but = sequence of mRNA
(with U for T)
Fig. 1
3-31
Polypeptides (proteins)
•
•
•
•
Amino acids joined together with peptide bonds
Chains of amino acids are polypeptides
Proteins are composed of 1 or more polypeptides
Polypeptides have polarity (as does DNA)
– Free amino group at one end is amino- (N-terminus)
– Free acid group at other end is carboxyl- (C-terminus)
Fig. 3
3-32
Protein Structure
• Proteins: polymers of amino acids linked through
peptide bonds
• Sequence of amino acids (primary structure) gives
rise to molecule’s:
– Local shape (secondary structure)
– Common types of secondary structure: H bonds of nearby
backbone
 a-helix
 b-sheet
– Overall shape (tertiary structure)
– Interaction with other polypeptides (quaternary
3-33
structure)
Secondary Structures - Tertiary structure
Figs. 4, 5
helix, pleated sheet
Fig. 6; myoglobin
Interaction of aa side
chains – longer range3-34
Protein Domains
• Compact structural regions of
protein are domains
• Immunoglobulins example of
4 globular domains (Fig. 8)
• Domains may contain
common structural-functional
motifs:
– Zinc finger
– Hydrophobic pocket
• Quaternary structure is
interaction of 2 or more
polypeptides
3-35
Relationship Between Genes and Proteins
one gene - one polypeptide hypothesis:
Most genes contain information for making 1 polypeptide
• 1902 suggestion link between disease alkaptonuria and
recessive gene
• If a single gene controlled production of an enzyme, lack of
enzyme could result in buildup of homogentisic acid, which is
excreted in urine
• If gene responsible for enzyme is defective, then enzyme ly
also is defective
• Many enzymes contain more than one polypeptide chain:
• Each polypeptide is usually encoded in one gene
3-36
mRNA is Information Carrier
• mRNAs carry genetic information from genes to
ribosomes, which synthesize polypeptides
• In 1950s and 1960s, concept of messenger RNA carries information from gene to ribosome:
• Intermediate carrier needed: in eukaryotes, DNA in
nucleus, proteins made in cytoplasm
• Jacob & Monod, from genetic experiments,
proposed ribosomes translate unstable RNAs called
messengers;
• Messengers are independent RNAs that move
information from genes to ribosomes
3-37
Transcription
• Transcription follows same base-pairing rules as
DNA replication
– U replaces T in RNA
– Base-pairing pattern ensures RNA transcript is
faithful copy of gene
• For transcription to occur at a significant rate,
reaction is enzyme-mediated
• RNA polymerase (RNAP) is enzyme directing
transcription
3-38
Synthesis of RNA
Fig. 20;
RNAP uses bp rules
3-39
Transcription
Phases
Initiation:
RNAP binds, local melting,
First few phosphodiester
(asymmetric synthesis)
Elongation:
RNAP links more
ribonucleotides 5’-> 3’
Termination:
RNAP, RNA and DNA
template dissociate
Fig. 14; much more detail later
3-40
Transcription Landmarks
• RNA sequences written 5’ to 3’, left to right
• Translation occurs 5’ to 3’; ribosomes reading
mRNA 5’ to 3’
• Genes written so that transcription proceeds from
left to right
• Gene’s promoter area lies just before start site,
said to be upstream of transcription
• Genes lie downstream of promoters
5’ _____P_____+1____ORF_____________ -3’
up
down
3-41
Translation on
Ribosomes
• Ribosomes are cell’s
protein factories
• Bacteria contain 70S
ribosomes (Euks 80S)
• Each ribosome has 2
subunits
– 50 S
– 30 S
• Each subunit contains
rRNA and many proteins
• No translation of rRNAs
Fig. 16
3-42
Transfer RNA: Adapter Molecule
• tRNA: small RNA recognizes
both mRNA and amino acids
• Cloverleaf model of tRNA
structure/function:
• One end (top, 3’ end) binds
specific amino acid
• Bottom end contains 3 bp
sequence (anticodon)that pairs
with complementary sequence
of mRNA (codon)
Fig. 17
3-43
Codons and Anticodons
• Enzymes that catalyze
attachment of amino acid
to tRNA are aminoacyltRNA synthetases
• A triplet in mRNA is codon
• Complementary sequence
to codon found in tRNA is
anticodon
Fig. 18
3-44
Initiation of Protein Synthesis
• Initiation codon (AUG) interacts with special
aminoacyl-tRNA
– In eukaryotes methionyl-tRNA
– In bacteria N-formylmethionyl-tRNA
• Position of AUG codon:
– At start of message AUG is initiator
– In middle of message AUG is regular methionine
• In Bacteria, Shine-Dalgarno sequence lies just
upstream of the AUG, functions to attract ribosomes
• Eukaryotes have special cap on 5’-end of mRNA;
ribosomes bind and find AUG
3-45
Translation
Elongation
• Initiating aminoacyl-tRNA
binds to P site on ribosome
• Amino acids added one at a
time to initiating amino acid
• First elongation step is
binding of second aminoacyltRNA to A site on ribosome:
• Process requires:
elongation factor, EF-Tu
Energy from GTP
Fig. 19
3-46
Termination of Translation; mRNA Structure
• 3 different codons (UAG, UAA, UGA) cause
translation termination
• Protein release factors recognize stop codons,
cause:
– Translation to stop
– Release of polypeptide chain
• Initiation codon and termination codon at ends
define an open reading frame (ORF)
3-47
**Structural Relationship Between Gene,
mRNA and Protein
Transcription of DNA does
not begin or end at same
places as translation
– Ex. Transcription
– begins at first G
– Translation begins
9-bp downstream
– This mRNA has
9-bp leader or
5’-untranslated region
5’-UTR
Fig. 20
3-48
Structural Relationship Between Gene,
mRNA and Protein, cont.
Trailer sequence is
present at 3’ end of
mRNA
– between stop codon
and transcription
termination site
– This mRNA has a
3’-untranslated region
or a 3’-UTR
3-49
3.3 Mutations
• Genes accumulate changes or mutations
• Mutation is essential for evolution
• If a nucleotide in a gene changes, likely a
corresponding change will occur in an amino acid of
that gene’s protein product
– If a mutation results in a different codon for the
same amino acid it is a silent mutation
– Often a new amino acid is structurally similar to
the old and the change is conservative
3-50
Example: Sickle Cell Disease
• Sickle cell disease genetic disorder (recessive)
• Disease results from single base change in gene for
b-globin (missense mutation: GAG -> GTG)
– insertion of incorrect amino acid into position of b-globin
protein (Glu -> Val)
– Altered protein distortion of red blood cells under lowoxygen conditions
• change in gene causes change in protein product
Fig. 25
3-51
Review questions Chapts. 2 and 3
2.3. Draw structure of phosphodiester bond, with flags for
bases, sugars clear, deoxy position indicated.
2.8 Draw principle of nucleic acid hybridization.
2AQ2 How many proteins of average size encoded by phage
T2 with 150,000 bp genome (assume no overlap)
3.2 Draw the structure of the peptide bond.
3.7 Describe the two main steps in gene expression.
3.12 How does tRNA serva as adaptor between the 3-bp
codons in mRNA and amino acids in proteins?
3.13-15 Consider single base mutations, and explain how they
could lead to premature termination of mRNA, to change
3-52
reading frame, or to substitute an amino acid.