1. 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

2. 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.

3. 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

4. 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

5. 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

6. 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

7. 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)

8. 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

9. 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

10. 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

11. 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

12. 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

15. 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:
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
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)

16. Bacterial Transformation – Griffith, 1928; Avery, 1944
DNA is Genetic Material
Bacterial Transformation – Griffith, 1928; Avery, 1944
Key experiments by Griffith in 1928:
Observed change in Streptococcus pneumoniae — from virulent (S) smooth colonies where bacteria had capsules, to avirulent (R) rough colonies without capsules
Heat-killed virulent cells could transform avirulent cells to virulent ones
In 1944 Avery confirmed, extended Griffith’s results, defined chemical nature of the transforming substance:
Techniques used excluded both protein and RNA as chemical agent of transformation
Other treatments verified that DNA is chemical agent of transformation of S. pneumoniae from avirulent to virulent
Fig. 2

17. 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

18. DNA is phage T2 genetic material Hershey – Chase 1952
Fig. 4
Phage T2

19. Purines and Pyrimidines
A and G are purines; C, T and U are pyrimidines
Note numbering of positions
Fig. 5

20. 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
Nucleosides lack phosphate
Nucleotides contain phosphate
Fig. 7

21. DNA nucleotide linkage
Nucleotides are nucleosides with phosphate group attached through phosphodiester bond
Nucleotides may contain 1, 2, or 3 phosphate groups
Fig. 9

22. 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

23. 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
Fig. 14

24. 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 double-stranded (M13, lambda)
Some viruses have RNA genes, either single- or double-stranded (MS2, HIV, rabies)

25. 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)

26. 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

27. 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

28. Phage DNA is typically circular; so are bacterial chromosomes
Some DNAs are linear – ex., eukaryotic chromosomes
Supercoiled DNA coils or wraps around itself like a twisted rubber band

29. 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

30. 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
Review Q: 1, 2, 4, 7, 9, 11, 12, 13*, 14*, 15*; AQ 1

31. 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

32. 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

33. 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 structure)

34. Secondary Structures - Tertiary structure
Figs. 4, 5
Fig. 6; myoglobin
helix, pleated sheet
Interaction of aa side chains – longer range

35. 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

36. 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

37. 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

38. 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

39. Synthesis of RNA
Fig. 20;
RNAP uses bp rules

40. Transcription Phases Initiation: (asymmetric synthesis) Elongation:
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

41. 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

42. 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

43. 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

44. Codons and Anticodons
Enzymes that catalyze attachment of amino acid to tRNA are aminoacyl-tRNA synthetases
A triplet in mRNA is codon
Complementary sequence to codon found in tRNA is anticodon
Fig. 18

45. 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

46. 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 aminoacyl-tRNA to A site on ribosome:
Process requires:
elongation factor, EF-Tu
Energy from GTP
Fig. 19

47. 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)

48. **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

49. 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

50. 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

51. 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 low-oxygen conditions
change in gene causes change in protein product
Fig. 25

52. 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?
Consider single base mutations, and explain how they could lead to premature termination of mRNA, to change reading frame, or to substitute an amino acid.