1. THE FLOW OF GENETIC INFORMATION LECTURES:
UNIT 1
THE FLOW OF GENETIC INFORMATION
LECTURES:
1. DNA Structure and Chemistry
2. Genomic DNA, Genes, Chromatin
3. DNA Replication, Mutation, Repair
4. RNA Structure and Transcription
5. Eukaryotic Transcriptional Regulation
6. RNA Processing
7. Protein Synthesis and the Genetic Code
8. Protein Synthesis and Protein Processing
Note: most slides have associated explanatary notes in this window.

2. THE FLOW OF GENETIC INFORMATION
DNA
RNA
PROTEIN 1 2 3
DNA
1. REPLICATION (DNA SYNTHESIS)
2. TRANSCRIPTION (RNA SYNTHESIS)
3. TRANSLATION (PROTEIN SYNTHESIS)

3. 1. DNA Structure and Chemistry
a). Evidence that DNA is the genetic information
i). DNA transformation
ii). Transgenic experiments
iii). Mutation alters phenotype
b). Structure of DNA
i). Structure of the bases, nucleosides, and nucleotides
ii). Structure of the DNA double helix
3’,5’-phosphodiester bond
polarity of the polynucleotide chains
hydrogen bonding of the bases
specificity of base pairing
complementarity of the DNA strands
c). Chemistry of DNA
i). Forces contributing to the stability of the double helix
ii). Denaturation of DNA
hyperchromicity
melting curves and Tm

4. Mice are injected either with Type R, non-virulent
i) DNA transformation: in vivo experiment
Mice are injected either with Type R, non-virulent
Streptococcus or with heat-killed, virulent Type S cells.
The DNA transformation experiments described in the next several slides show that DNA is the carrier of the
genetic information. The in vivo experiments show that something from the (heat-killed) virulent strain was able
to alter the (viable) non-virulent strain, converting some of the cells to virulent bacteria and killing the host. We
now know that purified DNA confers this virulence.

5. bacterial cells, making them lethal to the mice
Mice are injected with both Type R, non-virulent and
heat-killed, Type S Streptococcus
DNA carrying genes from the virulent, heat-killed cells transforms the non-virulent
bacterial cells, making them lethal to the mice

6. DNA transformation: in vitro experiment
Type R cells
Type R colonies
Type S cells
Type S colonies
This in vitro experiment shows that purified DNA from Type S cells is able to be taken up by Type R bacteria.
The process of getting functionally active DNA into cells is called DNA transformation. In this case,
transformation by Type S DNA altered the "genotype" of the host cells, since new genes were introduced into
these cells thus altering their genetic constitution. The expression of this Type S DNA changed the "phenotype"
of the transformed cells, making their colonies look "smooth" instead of "rough."
Type R cells
+ DNA from
Type S cells
Mixture of
Type R and Type S
colonies

7. Genotype: Phenotype: An organism’s genetic constitution.
The observed characteristics of an organism,
as determined by the genetic makeup
(and the environment).
DNA from Type S cells (thus conferring the Type S genotype)
transformed Type R cells into cells having the Type S phenotype

8. ii). Transgenic experiments
Injected into nucleus
of a fertilized mouse egg
Plasmid DNA carrying the
growth hormone gene
Egg implanted into uterus
of surrogate mother mouse
Transgenic experiments, which are usually carried out in mice, involve the transfer of a specific gene into the
nucleus of a fertilized egg. The gene integrates randomly into the chromosomal DNA and can be engineered to
be expressed in every cell, or only in certain cells at certain times. In this experiment, introduction of the
growth hormone gene into transgenic mice alters their genotype and confers the phenotype shown in the next
slide, which results from overexpression of growth hormone. Transgenic experiments show that specific
phenotypic traits can be conferred by specific genes, and thus that DNA is the carrier of the genetic information.
Other types of transgenic experiments involve mutation of specific genes in the mouse to determine the
functions of those genes and to create mouse models of human genetic disease. The mutation of a gene in a
transgenic mouse that eliminates the gene's function, is called a knockout mutation and the mouse carrying that
mutation is called a knockout mouse.
Mother mouse gives
birth to transgenic mouse

9. i). Structure of the bases, nucleosides, and nucleotides
b). Structure of DNA
i). Structure of the bases, nucleosides, and nucleotides
Purines
Pyrimidines
Adenine (A)
Thymine (T)
Be familiar with the structures of the purine bases, adenine (A) and guanine (G); and the pyrimidine bases,
thymine (T) and cytosine (C). A common base modification in DNA results from the methylation of cytosine,
giving rise to 5-methylcytosine (5mC). As we shall see in a subsequent lecture, 5mC is highly mutagenic.
5-Methylcytosine (5mC)
Guanine (G)
Cytosine (C)

10. [structure of deoxyadenosine]
When a base, such as adenine, is linked to a deoxyribose sugar through a glycosidic bond, the structure is a
nucleoside, in this case deoxyadenosine. The deoxyribose sugar lacks a hydroxyl group on the 2' carbon, hence
deoxy. This is in contrast to the presence of a hydroxyl at that position in the ribose sugar found in RNA. When
the deoxyribose sugar is phosphorylated, on either the 3' or the 5' position (or both), the structure is a nucleotide,
in this case deoxyadenosine-5'-phosphate. The precursors of DNA synthesis are
deoxynucleoside-5'-triphosphates or dNTPs.

11. Base +deoxyribose +phosphate
Nomenclature
Nucleoside Nucleotide
Base +deoxyribose phosphate
Purines
adenine adenosine
guanine guanosine
hypoxanthine inosine
Pyrimidines
thymine thymidine
cytosine cytidine
+ribose
uracil uridine
This table lists the common bases and their corresponding names when in the nucleoside or nucleotide form.
Hypoxanthine (inosine) is seen in DNA following deamination of adenine (adenosine). It is also seen in transfer
RNA as a common, functionally important posttranscriptional modification. Uracil (uridine) is found in RNA,
instead of thymine (thymidine), which is specific for DNA.

12. 3’,5’-phosphodiester bond
ii). Structure of the
DNA double helix
The polynucleotide chain is formed by linking nucleotides through 3',5'-phosphodiester bonds.
polynucleotide chain
3’,5’-phosphodiester bond

13. hydrogen bonding of the bases
A-T base pair
The DNA double helix requires that the two polynucleotide chains be base-paired to each other. This slide
shows an adenine-thymine (A-T) base pair (which is the A and which is the T?); and a guanine-cytosine (G-C)
base pair (which is the G and which is the C?). Because of base pairing, the polynucleotide chains in
double-stranded DNA are complementary to each other.
G-C base pair
Chargaff’s rule: The content of A equals the content of T,
and the content of G equals the content of C
in double-stranded DNA from any species

14. specificity of base pairing complementarity of the DNA strands
Double-stranded DNA
This slide shows double-stranded DNA, which is composed of two base-paired, complementary polynucleotide
chains. As shown on the next several slides, base pairing is required for two important functions of DNA: 1) DNA
replication involves an unwinding of the double helix followed by synthesis of a complementary strand from
each of the unpaired template strands, and 2) DNA serves as a template for RNA synthesis by utilizing the
information in one strand to code for a complementary RNA strand.
DNA in the "B" form has a major groove and a minor groove, and has 10 base pairs per one turn of the double
helix. DNA that is overwound or underwound, with fewer than or more than 10 base pairs per turn, is said to be
"supercoiled". It should also be noted that the complementary strands in double helical DNA are antiparallel with
respect to each other.
specificity of base pairing
complementarity of the DNA strands
B-DNA has 10 base-pairs per turn

15. base pairing during DNA synthesis
Parental DNA strands
Daughter DNA strands
base pairing during RNA synthesis

16. DNA that is over- or underwound is “supercoiled”
positive supercoiling results from overwinding DNA
and normally occurs during DNA replication
negative supercoiling results from underwinding DNA
and normally occurs in the nucleosome
negative supercoiling can give rise to Z-DNA
Z-DNA is a left handed helix
with zigzagged (hence Z) phosphates
Z-DNA occurs where there are
alternating pyrimidines and purines (on one strand)
the transition of B- to Z-DNA is
facilitated by 5-methylcytosine
negative supercoiling may affect RNA synthesis
by promoting Z-DNA formation
by making it easier to separate the DNA strands

17. The structure of Z-DNA is shown on the left in comparison to B-DNA on the right. Z-DNA has a left-handed helix,
whereas B-DNA has a right-handed helix. In addition, Z-DNA is elongated and thinner, and the major groove is
flattened out on the surface of the helix. The phosphate backbone of Z-DNA zigzags (hence, Z). Regions of DNA
that contain alternating pyrimidines and purines on one strand are more likely to adopt a Z-DNA conformation.
The transition is also facilitated by the presence of 5-methylcytosine and by negative supercoiling. DNA is a
dynamic molecule and therefore moves from one conformation to another based on external forces in the cell. It
is likely that B- to Z- transitions are involved in the regulation of gene expression.

18. antiparallel polarity of the polynucleotide chains
5’carbon 5’ 3’
3’carbon
antiparallel polarity of the
polynucleotide chains
Each polynucleotide chain has a 5' end and a 3' end. In the DNA double helix, the strands are arranged in an
antiparallel fashion, that is with opposite polarity with respect to each other. 3’ 5’

19. nucleases hydrolyze phosphodiester bonds
Exonucleases cleave at
terminal nucleotides. 5’ 3’
e.g., proofreading exonucleases
Endonucleases cleave internally
and can cut on either side of a
phosphate leaving 5’ phosphate
or 3’ phosphate ends depending
on the particular endonuclease.
Deoxyribonucleases (or DNases) are enzymes that cleave phosphodiester bonds. Some are used for
constructive purposes, such as proofreading during DNA replication, whereas others are used to degrade DNA.
There are two basic classes of DNases: exonucleases and endonucleases. Exonucleases remove only the
terminal nucleotide, whereas endonucleases cleave anywhere within the DNA double helix.
e.g., restriction endonucleases 3’ 5’

20. c). Chemistry of DNA
i). Forces affecting the stability of the DNA double helix
hydrophobic interactions - stabilize
- hydrophobic inside and hydrophilic outside
stacking interactions - stabilize
- relatively weak but additive van der Waals forces
hydrogen bonding - stabilize
- relatively weak but additive and facilitates stacking
electrostatic interactions - destabilize
- contributed primarily by the (negative) phosphates
- affect intrastrand and interstrand interactions
- repulsion can be neutralized with positive charges
(e.g., positively charged Na+ ions or proteins)
Three types of forces contribute to maintaining the stability of the DNA double helix: 1) hydrophobic interactions, 2) stacking interactions, and 3) hydrogen bonding. The base pairs in the interior of the DNA molecule create a hydrophobic environment, with the negatively charged phosphates along the backbone being exposed to the solvent. Thus, in an aqueous environment, the double-stranded structure is stabilized by the hydrophobic interior. Reagents that solubilize the DNA bases (e.g., methanol) destabilize the double helix. Stacking interactions and hydrogen bonding interactions are relatively weak but additive. Reagents that disrupt hydrogen bonding [e.g., formamide, urea, and solutions with very low pH (pH <2.3) or very high pH (pH >10)] destabilize the double helix.
Electrostatic replusion by negatively charged phosphates along the DNA backbone destabilize the double helix. For example, if the phosphates are left unshielded, as when DNA is dissolved in distilled water, the DNA strands will separate at room temperature. Neutralizing these negative charges by the addition of NaCl (which contributes positively charged sodium ions) to the DNA solution will prevent strand separation. In the cell, the phosphates also interact with positively charged magnesium ions and with positively charged (basic) proteins.

21. Hydrophilic phosphates5’ 3’
Hydrophilic phosphates
Hydrophilic phosphates
This slide shows the hydrophobic interior and the hydrophilic exterior of the DNA double helix. 3’
Hydrophobic
core region 5’

22. Stacking interactions
Charge repulsion
This slide shows base stacking and charge repulsion in the DNA double helix.
Charge repulsion

23. Model of double-stranded DNA showing three base pairs
This slide shows a side view of three base pairs in the DNA double helix. Note the base-pair stacking
interactions, the hydrophobic interior, and the phosphates on the exterior.
Model of double-stranded DNA showing three base pairs

24. ii). Denaturation of DNA
Strand separation
and formation of
single-stranded
random coils
Double-stranded DNA
Extremes in pH or
high temperature
A-T rich regions
denature first
The forces stabilizing the DNA double helix can be overcome by heating the DNA in solution or by treating it
with very high or very low pH (low pH will also damage the DNA, whereas high pH will simply separate the
polynucleotide chains). When the strands of DNA separate, the DNA is said to be denatured (when high
temperature is used to denature DNA, the DNA is said to be melted). Because some of the forces stabilizing the
DNA double helix are contributed by base pairing interactions, and because A-T base pairs have only two
hydrogen bonds in contrast to G-C base pairs which have three hydrogen bonds, regions of the DNA duplex that
are A-T rich will denature first. Once denaturation has begun, there is a cooperative unwinding of the double
helix that ultimately results in complete strand separation.
Cooperative unwinding
of the DNA strands

25. Electron micrograph of partially melted DNA
Double-stranded, G-C rich
DNA has not yet melted
A-T rich region of DNA
has melted into a
single-stranded bubble
This slide shows an electron micrograph tracing of a DNA molecule that is only partially melted. The thicker
regions are double-stranded and probably more G-C rich. The A-T rich regions are more prone to denaturation,
and as seen here, form single-stranded "bubbles."
A-T rich regions melt first, followed by G-C rich regions

26. hyperchromicity Single-stranded Double-stranded Absorbance 220 260 300
When a solution of double-stranded DNA is placed in a spectrophotometer cuvette and the absorbance of the
DNA is determined across the electromagnetic spectrum, it characteristically shows an absorbance maximum at
260nm (in the UV region of the spectrum). If the same DNA solution is melted, the absorbance at 260nm
increases approximately 40%. This property is termed "hyperchromicity." The hyperchromic shift is due to the
fact that unstacked bases absorb more light than stacked bases.
220
260
300
The absorbance at 260 nm of a DNA solution increases
when the double helix is melted into single strands.

27. 100 50 70 90
Temperature oC
Percent hyperchromicity
DNA melting curve
Tm is the temperature at the midpoint of the transition
Hyperchromicity can be used to follow the denaturation of DNA as a function of increasing temperature. As the
temperature of a DNA solution gradually rises above 50 degrees C, the A-T regions will melt first giving rise to an
increase in the UV absorbance. As the temperature increases further, more of the DNA will become
single-stranded, further increasing the UV absorbance, until the DNA is fully denatured above 90 degrees C. The
temperature at the mid-point of the melting curve is termed "melting temperature" and is abbreviated Tm. The
Tm for a DNA depends on its average G+C content: the higher the G+C content, the higher the Tm. Note: G+C
content, G-C content, and GC content are equivalent terms.

28. average base composition (G-C content) can be
determined from the melting temperature of DNA 50 70 60 80
Temperature oC
Tm is dependent on the G-C content of the DNA
Percent hyperchromicity
E. coli DNA,
which is 50% G-C,
has a Tm of 69 o C.
This slide shows the dependence of Tm on average G+C content of three different DNAs. Under the conditions
used in this experiment, E. coli DNA which has an average G+C content of about 50%, melted with a Tm of 69
degrees C. The curve on the left represents a DNA with a lower G+C content and the curve on the right
represents a DNA with a higher G+C content. Tm is dependent on the ionic strength of the solution. At a fixed
ionic strength there is a linear relation between Tm and G+C content. For example, at 0.2M sodium ion
concentration, Tm = (%G+C). Therefore, a DNA that is 50% G+C will melt at 89.8 degrees C in 0.2M
sodium ion.