DNA

1. BME 403: Molecular Biology for Engineers
The Central Dogma, DNA, RNA
Lecture 1
Akid Ornob

2. Objective - The Central Dogma

3. Cells – Basic unit of life
• Cells – basic unit of a living organism
• It contains hereditary information which it passes on
to the next generation
• Cells gather raw materials from its environment
• And takes decisions through a complex networks of
chemical reactions known as pathways
• > 10^13 cells in an average human

4. Cells
Prokaryotic cell Eukaryotic cell
1. Eukaryotic cells have nucleus that encapsulates their DNA
2. Prokaryotic cell do not have nucleus
3. Eukaryotic cells contain membrane-bound organelles

5. Nucleus and DNA packaging
Chromatin carries DNA and histone protein

6. 6
Adenosine Mono Phosphate - AMP
-H in DNA
base
sugar
phosphate
AMP is a nucleotide building block (dAMP for DNA/RNA)
The structure of nucleic acids (DNA/RNA)

7. 7
Uracil in RNA
Bases for DNA are shown above. RNA has uracil instead of thymine.
Nucleotides are - for RNA or DNA
Adenosine Guanosine Uridine Cytidine
Deoxyadenosine Deoxyguanosine Deoxythymidine Deoxycytidine
The chemical nature of polynucleotide

8. Nucleosides
Carbon numbering of sugars in nucleosides is important

9. • Ordinary numbers are used in bases
• Primed numbers are used sugars
• Base is linked to 1’ position
• 2’ position is deoxy in deoxynucleosides
• Sugars are linked together in DNA and RNA through their 3’
and 5’ -positions
Carbon numbering in nucleosides

10. Nucleotides (Sugar + Base +
phosphate)
• A nucleotide is a nucleoside with a phosphate group attached
through a phosphodiester bond

11. 11
The phosphodiester bond

12. 12
Watson-Crick
basepairing
scheme
Hoogstein
basepair
scheme
Protonated C
+
Base-pairing in DNA
In double-stranded
DNA:
A always bonds to T
C always bonds to G
The bases are held
by hydrogen
bonding
- 2 H bonds
between A and T
- 3 H bonds
between C and G

13. DNA as a double helix
Exists as a double helix
2 strands of DNA
running anti-parallel to
each other (one 5’-3’
and the other 3’-5’)
The DNA sequence is
read from 5’-3’

14. DNA replication
DNA replication is semi-conservative

15. DNA replication – interplay between
enzymes
DNA replication occurs prior to cell division and is essential
transferring the genetic information to the next generation

16. Mutation
• Accuracy of DNA replication is extraordinary
• Several proofreading mechanisms are used
• DNA is copied with fewer than one mistake in 109 nucleotides added
• Very rarely, the replication skips or adds a few nucleotides, or puts a
T instead of C or A instead of G
• Any change of this kind is a genetic mistake, call a mutation.

17. Effect of Mutation
• Mutation cause alteration of gene
• May lead to inactivation of a crucial protein
• Result in cell death, mutation will be lost
• Mutation may be silent
• Very rarely, improved gene with novel function
• In this case mutation will be carried to next generation
• May lead to uncontrolled cell division and the formation of a tumor
• Tumor may be malignant and cause cancer

18. DNA Repair
• The altered portion is recognized
and removed by enzymes called
DNA repair nucleases

19. The chromosome
• DNA is packaged into individual
chromosomes
• Prokaryotes typically have a large single
chromosome (e.g. bacteria, archea)
• Eukaryotes have species specific number of
linear chromosomes (e.g. animals, plants)
• A growing field of research in genetics and
bioinformatics is epigenetics – interaction of
the genetic code with the histone protein
and its effects on the phenotype of a cell

20. Gene and the genome
• Genome – complete set of DNA for a given
species
• Human genome -> 23 pairs of chromosomes
• All cells except for the sex cells and mature
red blood cells contain the complete
genome
• Genes – basic unit of heredity
• Contains a sequence of nucleic acid bases
that encodes the information required to
synthesize a particular protein
• Human genome consists of ~25,000 protein
coding genes

21. Gene density
Not all of the DNA in a genome encodes protein
Bacteria – 90% coding gene/kb
Human – 1.5% coding gene/kb (Total genetic material = 3Gb)
• Eukaryotic genes are broken into pieces
called exons
• Separated by seemingly meaningless
piece called introns
• Prokaryotic genes are continuous

22. Gene Sequence Determines Protein
Amino Acid Sequence

23. 23
Sugar (ribose) Pucker
The critical difference in RNA and
DNA double helix
3’ -OH in RNA stabilizes C-3’ endo
ribose configuration
RNA
B-DNA

24. 24
Sugar Pucker
The sugar ring has different
conformations for RNA and
DNA
Note the locations and distances of
phosphate groups in RNA and DNA
differ due to change in sugar ring
conformation
RNA
B-DNA
B-DNA RNA (A-DNA)
Substantial consequences
Backbone is more extended
In DNA

25. 25
Double-stranded DNA structure
A. Initially deduced from low resolution fiber diffraction studies (X-ray on fibers)
B. High resolution structure from crystals of short DNA oligomers
-much more detail
-base pair variations, structural variations (B-DNA, A-DNA, Z-DNA)

26. 26
Double-strand DNA/RNA Fiber Diffraction Studies: 3 major
families
B-family A-family Z-family
B RNA left handed helix
C RNA•DNA
D DNA
B-DNA: standard form of double helix of DNA
A-DNA: induced by binding of eukaryotic transcriptional
promoter (TATA binding protein) to DNA
Z-DNA: May form in regions of the genome with dinucleotide repeats
(CA)n or (CG)n

27. 27

28. 28
DNA is polymorphic-environmental
conditions can alter structure
“Phase diagram” relating conformational changes of natural DNAs with
different (dG+dC) content with varying relative humidity.

29. 29
A-DNA is like
double-stranded RNA
B-DNA is the
predominant form
of double-strand DNA

30. 30

31. 31
The phosphate groups of A-DNA and Z-DNA can be “economically”
hydrated by a single bridging water - this is why these DNA forms are
favored at low hydration
High salt also favors Z-DNA based on electrostatics from phosphate-repulsion
(PNAS 1993 90, 5740)
Comparison of deoxyribose-3’, 5’-diphosphate
fragments in a, A-, b, B- and c, Z-DNA. Vertical
lines represent helical axes; phosphorous atoms
indicated by shading, phosphate oxygens by
numbering. Shortest distances between free
phosphate oxygen atoms are give in Å, W
represents water molecules that bridge free
phosphate oxygens in A- and Z-DNA. Capital
letters B in A- and B-DNA and G, C in Z-DNA
indicate C(1’) sugar atoms where bases are
attached
Nature 1986 324, 385-
A-DNA
B-DNA
Z-DNA

32. 32
Z-DNA-left handed helix
Defined by crystal structure of d(CpGpCpGpCpG)
-12 bp/turn
-alternating syn anti base configuration
-major groove “filled” (only minor groove)
Can be formed when (GC)n tracts are present in DNA sequence
Methylation of cytosine favors
B Z
Biological relevance???
Still not clear. May
be present in stretches of
repeating dinucleotides with
alternating
purine/pyrimidine

33. 33
Z-DNA vs B-DNA
CpGpCpGpCpG (left handed) (right handed)
Van der Waals side views of Z-DNA and B-DNA. Two views of Z-DNA are shown which are
30o apart in orientation about the helix axis.
Zig-Zag
backbone
Z-DNA
B-DNA

34. 34
dsDNA reversibly self-assembles at physiological temperature, pressure, pH, and
ionic composition: Gds < Gss

35. 35
Thermal Denaturation of dsDNA

36. DNA vs RNA
• Genetic information encoded in a
particular gene (strand of DNA) is
transferred to RNA via transcription
• RNA retains all the information of DNA
sequence
• However the differences are
• RNA contains ribose instead of
deoxyribose sugar
• The base thymine (T) is replaced by
uracil (U)
• RNA is usually single stranded and
structurally more versatile than DNA

37. • Ribonucleic acids play three well-understood roles in living
cells:
• Messenger RNAs encode the amino acid sequences of all the
polypeptides found in the cell
• Transfer RNAs match specific amino acids to triplet codons in mRNA
during protein synthesis
• Ribosomal RNAs are the constituents of ribosomes and catalyzes
protein synthesis
• Ribonucleic acids play several less-understood functions in
eukaryotic cells:
• MicroRNA appears to regulate the expression of genes, possibly via
binding to specific nucleotide sequences
• Ribonucleic acids act as genomic material in viruses
Overview of RNA function

38. • Ribonucleic acids are synthesized in cells using DNA as a
template in transcription
• Transcription is tightly regulated in order to control the concentration
of each protein
• Being mainly single-stranded, many RNA molecules can fold
into compact structures with specific functions
• Some RNA molecules can act as catalysts (ribozymes), often using
metal ions as cofactors
• Most eukaryotic ribonucleic acids are processed after
synthesis
• Elimination of introns; joining of exons
• Poly-adenylation of the 3’ end
• Capping the 5’ end
Overview of RNA metabolism

39. Initiation:
- RNA polymerase binds to sequence called promoter to
begin transcription. DNA double helix unwinds
- RNA polymerase covers about a 35 bp-long segment of DNA
Elongation:
- RNA polymerase unwinds from the promoter region and
adds nucleoside triphosphates add to the 3’ end of the
growing RNA strand
- The growing chain is complementary to the template strand
of the DNA
Termination:
- RNA transcription ends when the RNA Polymerase
encounters a terminating signal in the DNA -> formation of
an RNA “hairpin” structure which destabilizes the
interaction between the Polymerase and the RNA
- The transcribed mRNA is released
Mechanism of transcription in E.Coli

40. Detailed Mechanism

41. Template and Coding strands
• DNA Template Strand – serves as template
for RNA polymerase
• DNA Coding Strand – the non-template
strand; has the same sequence as the RNA
transcript

42. mRNA processing
• Introns cleaved
• Exons spliced
together
• Poly-A cap on the 3’
end (stabilizes the
mature mRNA
structure)

43. Translation and Protein Biosynthesis
Protein life cycle in eukaryotes

44. • There are 20 common, genetically encoded amino
acids
• A four-letter code in groups of two is insufficient
(16)
• A four-letter code in groups of three IS sufficient (64)
• Living organisms use non-overlapping mRNA code
with no punctuation
The genetic code for proteins consists of
triplets of nucleotides

45. Overlapping vs Non-overlapping code

46. Reading Frames
• Reading frame is set at the initiation of translation process
• In almost every case, only one reading frames will produce a functional protein
• This reading frame is known as open reading frame

47. • The code is written in the 5’ → 3’ direction
• Third base is less important in binding to tRNA
• First codon establishes the reading frame
• If reading frame is thrown off by a base or two, all
subsequent codons are out of order
• 61/64 codons code for amino acids
• Three are termination codons
• UAA, UGA, UAG
• AUG = initiation codon (as well as Met codon)
Features of the genetic code

48. Nucleotide Code Dictionary

49. Genetic code is degenerate
• 4 nucleotides; 43=64 codons possible
• 3 sequences for stop codon, 1 for start (also
Met)
• 61 different codons to specify only 20 amino
acids
• Only Met and Trp have unique codons
• Either each amino acid is specified by >1
tRNA or tRNAs can bind to more than one
codon (Both occur)

50. Genetic Code
• Amino acid sequence of protein is constructed
through the translation of information encoded
in mRNA
• Amino acids are specified by mRNA codons
consisting of nucleotide triplets. Translation
requires adapter molecules, the tRNAs, that
recognize codons and insert amino acids at
the correct position in the protein

51. Genetic code (cont.)
• AUG signals initiation, and UAA, UAG, and UGA signal
termination
• Genetic code is degenerate—multiple code words for almost
every amino acid
• Standard genetic code words are universal for almost every
species
• Third position in each codon is less specific than the first two

53. Adapter (tRNA) brings amino acid to mRNA

54. • The codon sequence is complementary with the
anticodon sequence
• The codon in mRNA base pairs with the anticodon in
mRNA via hydrogen bonding
• The alignment of two RNA segments is antiparallel
Molecular Recognition of Codons in mRNA
by tRNA

55. • ssRNA of 73–93 nucleotides in
both bacteria and eukaryotes
• Cloverleaf structure in 2-D
• “Twisted L” shape in 3-D
• Most have G at 5’-end; all have
CAA at 3’-end
• Amino acid arm
• Has amino acid esterified via
carboxyl group to the 2’-OH or
3’-OH of the A of the terminal
CAA codon
Structure and characteristic features of
tRNA

56. • Anticodon arm
Have modified bases
Methylated bases, etc.
• D arm
Contains dihydrouridine (D)
Contributes to folding
• TψC arm
Contains pseudouridine (ψ)―has bonding
between base and ribose
Helps in folding
Structure and characteristic features of
tRNA

57. 3D structure of yeast tRNA by X-ray
Diffraction

58. • When different codons specify one amino acid, the difference lies at the
third position of the codon (5’ end of tRNA anticodon)
• Wobble or flexibility at this site allows the tRNA anti-codon to read multiple
codon sequences
• Minimum of 32 tRNAs are required to translate all 61 codons (31 for amino
acids and 1 for initiation with fMet)
Wobble base-pairing in anticodon

59. “Wobble” pairings in tRNA with
mRNA can occur in the third base
• The third base of a codon can form non-canonical base
pairs with its complement (anticodon) in tRNA
• Some tRNAs contain Inosinate (I), which can H-bond
with U,C, and A
• These H-bonds are weaker and were named by Crick as
“wobble” base pairs
• Example: In yeast, CGA, GCU, and CGC all bind to tRNAArg,
which has the anticodon 3’-GCI-5’
• Although sequences are usually written 5’🡪3’, the anticodon here is
written 3’🡪5’ to illustrate its bonding to the mRNA codons

60. “Wobble” pairings in bacteria and
eukaryotes

61. Anticodon containing inosinate (G or A)
can pair with any of 3 codon sequences

62. Ribosome and polypeptide
synthesis
• Ribosome is a large protein synthesis machine
• It finds the specific start site that sets the reading frame
• As the ribosome moves along the mRNA, it translates codons to amino acids using
tRNA
• At the end, stop codon, it releases protein to cytoplasm and detach itself
• 2 Subunits of ribosome
• “Small” subunit involved in the recognition of
the mRNA and tRNA sequences
• “Large” subunit attaches the amino acid to
form a polypeptide chain

63. 1) Activation of amino acids
• tRNA is aminoacylated
2) Initiation of translation
• mRNA and aminoacylated tRNA bind to ribosome
3) Elongation
• Cycles of aminoacyl-tRNA binding and peptide bond
formation…until a STOP codon is reached
4) Termination and ribosome recycling
• mRNA and protein dissociate, ribosome recycled
5) Folding and post-translational processing
• Catalyzed by a variety of enzymes
Five stages of protein synthesis

64. Overview of protein synthesis

65. Additional reading – translation

66. Additional reading – mechanism of translation

67. Next Class – Analyzing and Sequencing Nucleic
Acids
• Copying DNA
• Amplifying DNA
• Probing DNA
• Measuring DNA length
• Sequencing DNA

68. Thank You