test

1. DNA Replication and Protein
Synthesis
Patricia Caldani MS

3. Objective
Genome structure
Chromosome/Gene/DNA
DNA Replication
Protein Synthesis
Transcription and Translation
Mutations

4. The Human Genome
The human genome is made up of 3 x
9
10 base pairs of DNA (haploid
genome)
This contains 30,000 genes arranged
on 46 chromosomes
Packaged within the nucleus of the
cell

5. What is a Gene??
Genes are instruction manuals for our
bodies
They are the directions for building
all the proteins that make our body
function
Genes are made up of DNA
EXP. RBC use Hemoglobin

6. Chromosomes
 Long strands of DNA packaged and
compressed very tightly
 Everyone has __23_________ copies
of each chromosome
 1 pair of each of the 22 ‘autosomes’ <22>
• plus XX for a female (46XX) <1>
• or XY for a male (46XY) <OR 1>
 DIPLOID GENOME

8. Chromosomes in
Metaphase
Telomere
Short arm (p)
Centromere
Long arm (q)
Telomere

9. DNA (DeoxyriboNucleic Acid)
2 major functions
 Direction of all protein synthesis
 Accurate transmission of this information from one
generation to the next
Fundamental to
 Cell metabolism
 Cell division

10. DNA (DeoxyriboNucleic Acid)
 String of deoxyribose
sugars joined by
_____phosphate______
groups.
 Each sugar is attached to
one of 4 possible
nucleotide bases
 ADENINE (A),
 CYTOSINE (C ),
 GUANINE (G) or
 THYMINE (T)

11.  Each hydrogen bond
hold two DNA strands
together very tightly.
 To form Watson-Crick
base pairs, DNA strands
must be anti parallel &
____double
stranded_______

12. DNA
Double helix structure
2 strands are held together by
hydrogen bonds
4 bases pairing rule
o Adenine = Thymine (A = T)
o Guanine = Cytosine (G = C)

13. DNA Base Pairing
A G C G A T C T G G
T C G C T A G A C
C
Double helix consists of 2 complimentary strands of
DNA.

14. Some Definitions
Replicon: Unit in which replication occurs
Origin: Position at which replication initiates
Terminus: Position at which replication terminates
Origin Replicon
bo naA h
sit pera s
rep gene
O ron
D -ric
r
to
Ite
AT
s
xe
e

16. DNA Replication is Semiconservative
3’
5’
Semi-
5’
3’
Conservative conservative
Dispersive

17. Semiconservative Replication of Density-Labeled DNA
DNA replication is
semi-conservative: in
the "next generation"
molecule one strand
is "old" and another
is "new"

18. DNA Replication
Semi – conservative replication
C
T ATC
G
A A TAG
ATC T
TAG
TA
DNA AT ATC
C TAG
unzips G
Original double strand
DNA separates and replicates 2 new double strands
– each containing one parent
and one daughter strand

19. Many enzymes are involved in
DNA replication

20. The Enzymes of DNA
Replication
 1. Topoisomerase - initiates the unwinding of the super coiled DNA by
clipping the DNA backbone.
 2. Helicase - separates the double strand by "melting" the hydrogen
bonds that hold the bases together. It requires energy (in the form of
ATP ).
 3. Primase - makes a short RNA segment (called a primer) that is
complementary to the DNA strand at specific sites. It "primes" for
DNA replication because it provides an - OH for DNA polymerase to
attach the first DNA nucleotide. The RNA primer is later removed and
the gap is filled in with DNA nucleotides.
 4. DNA polymerase (III- major polymerase) - binds to one side of the
DNA and nucleotides to bond with their complementary pair
 5. Ligase – joins two large molecules via covalent bonds-repair
discontinued SS-DNA strands

21. Common Features of Replication
Origin
 Unique DNA sequence containing multiple
short repeated sequence
 Origins contain DNA sequences recognized by
replication initiator proteins
 AT-rich stretch

22. DNA replication starts within
a special region of DNA
called REPLICATION
ORIGIN which is defined by
a specific nucleotide sequence
Replication of DNA is
____semiconservative_
______
Two Y-shaped replication
forks are moving in the
opposite directions during
DNA replication

23. Initiation of DNA Replication
 DnaB is a helicase.
 An enzyme moves
along DNA duplex
utilizing the energy of
ATP hydrolysis to
separate the strands.
 5’-3’
 SSB: single strand
binding protein.

24. DNA synthesis always starts
with a RNA primer

25. The Role of RNA
Primer in DNA
Replication
 E. coli primase catalyze
the RNA Primer for
DNA Synthesis
 dnaG
 Primer: <15 nts

26. DNA Polymerase
 Unable to separate the two strands of DNA
 Only elongate a pre-existing DNA or RNA
(Primer)
 Only add nucleotides to the 3’-hydorxyl group,
i.e., only 5’-3’ synthesis

27. DNA Synthesis Occurs in the 5’→3’ Direction
P P P P OH 3’
5’
3’ 5’
P P P P P P P P P
Incoming nuceolotide
triphosphate
5’PPP OH3’
5’ P P P P OH 3’
3’ 5’
P P P P P P P P P
Nucleotide monophosphate
added to chain with release
of diphosphate PP
5’ P P P P P OH 3’
3’ 5’
P P P P P P P P

28. DNA Synthesis is Semidiscontinous
Lagging strand synthesis
5’
3’ 3’
5’ 5’
3’
Leading strand synthesis

29. DNA Polymerase Exonuclease
Activity
The enzyme that synthesizes DNA
is
______POLYMERASE________
__________ self-correcting:: it has
a proofreading activity

30. It is not trivial to replicate both DNA
strands in the 5' to 3' direction!

31. Growing Fork
 Leading
strand:
continuous
 Lagging
strand:
discontinuous

32. Lagging strand synthesis
 Leading strand:
continuous
 Lagging strand:
discontinuous
 Okazaki fragment:

33. Gap
Removal
 Pol I
 5’-3’
exonuclease
 Fills gap
DNA Pol I
-exonuclease- reads the
fragments and removes
the RNA Primers.
 Ligase

34. Ligase
DNA Ligase: adds
phosphate in the
remaining gaps of the
phosphate - sugar
backbone

35. Proofreading by 3’ to 5’
Exonuclease

36. Proofreading by 3’ to 5’
Exonuclease activity of DNA Pol

37. Final Step-Termination
 This process happens when the DNA
Polymerase (enzymes) reaches to an end of both
strands.
 The end of the parental strand where the last
enzymes binds aren't replicated.
 These ends of chromosomal DNA consists of
noncoding DNA.

39. From Genes to
Proteins
Patricia Caldani MS

40. DNA and RNA differ in 3 ways
RNA DNA
 Single-stranded  Double-stranded
 Ribose (sugar)  Deoxiribose (sugar)
 Uracil (base)  bonds to  Thymine (base) 
Adenine bonds to Adenine

41. The Genetic Code
 Every three bases of DNA is called a ‘codon’
 Each codon specifies an amino acid
 Codons specify amino acid sequence of
protein

42. Amino Acid Code
•64 possible triplet codons
•Only 20 amino acids
•Code is “degenerate or redundant”
Alani ne Ala A Leuci ne Leu L
Argi ni ne Arg R Lysi ne Lys K
Asparagi ne Asn N Methi oni ne Met M
Asparti c Aci d Asp D Phenylalani ne Phe F
Cystei ne Cys C Proli ne Pro P
Glutami ne Gln Q Seri ne Ser S
Glutami c Aci d Glu E Threoni ne Thr T
Glyci ne Gly G Tryptophan Trp W
Hi sti di ne Hi s H Tyrosi ne Tyr Y
Isoleuci ne Ile I Vali ne Val V

43. Genetic Code
U URACIL C CYTOCINE A ADENINE G GUANINE
U UUU Phe (F) CUU Leu (L) AUU Ile [I] GUU Val [V] U
UUC Phe (F) CUC Leu (L) AUC Ile [I] GUC Val [V] C
UUA Leu (L) CUA Leu (L) AUA Ile [I] GUA Val [V] A
UUG Leu (L) CUG Leu (L) AUG Met [M] GUG Val [V] G
C UCU Ser (S) CCU Pro [P] ACU Thr [T] GCU Ala [A] U
UCC Ser (S) CCC Pro [P] ACC Thr [T] GCC Ala [A] C
UCA Ser (S) CCA Pro [P] ACA Thr [T] GCA Ala [A] A
UCG Ser (S) CCG Pro [P] ACG Thr [T] GCG Ala [A] G
A UAU Tyr [Y] CAU His [H] AAU Asn [N] GAU Asp [D] U
UAC Tyr [Y] CAC His [H] AAC Asn [N] GAC Asp [D] C
UAA Ter [end] CAA Gln [Q] AAA Lys [K] GAA Glu [E] A
UAG Ter [end] CAG Gln [Q] AAG Lys [K] GAG Glu [E] G
G UGU Cys [C] CGU Arg [R] AGU Ser [S] GGU Gly [G] U
UGC Cys [C] CGC Arg [R] AGC Ser [S] GGC Gly [G] C
UGA Ter [end] CGA Arg [R] AGA Arg [R] GGA Gly [G] A
UGG Trp [W] CGG Arg [R] AGG Arg [R] GGG Gly [G] G

44. Gene Structure
Promoter Exons Introns
AUG UAA
start UAG ‘stop’
UGA
Exon = coding sequence
Intron= intervening sequence (non-coding)

45. Protein Synthesis
Messenger - RNA
DNA Code

46. The Flow of Information

47. Protein Synthesis
transcription
DNA RNA Protein
translation

48. The Flow of Information

49. Transcription
 Double DNA strands separate
 DNA sense strand acts as template and is
‘transcribed’ into messenger RNA (mirror image
of the DNA but Uracil instead of Thymine)
 Introns are sliced out of the sequence
DNA ATCGG
UAGCC
mRNA

50. Transcription
 This is the first step in Protein Synthesis:
1. The instructions are ______________
(“transcribed”) to an RNA molecule.

51.  To sum up Transcription…
 Info transferred from DNA to RNA
 What is the Enzyme involved in Transcription?
 Answer  RNA Polymerase

52. Transcription has 3 steps…
1 – RNA
Polymerase
binds to the
gene’s promoter
(DNA)  (like
a starting line in
a race).

53. 2 – RNA Polymerase UNWINDS the DNA
molecule. The DNA nucleotides are exposed.

54. 3 – RNA Polymerase adds complimentary
______________ to separated DNA strand.
** Remember  RNA has ______________
instead of Thymine for a base.

55.  The RNA Polymerase will continue
transcription until it reaches the “stop signal” on
the DNA molecule (like a finish line).
 Then the RNA strand is released and goes on to
the next step…Translation

56. Transcription

57. Transcription

58. Transcription
RNA
polymerase
exon 1 2 3
intron 1 2
transcription
factors
5’ 3’

59. Transcription
RNA
polymerase
exon 1 2 3
intron 1 2
transcription
factors
5’ 3’

60. Transcription
5’GGAUUCGUGCUGCUAA
RNA
polymerase
exon 1 2 3
intron 1 2
transcription
factors
5’GGATTCGTGCTGCTAA

61. Transcription
primary
transcript
exon 1 2 3 RNA
polymerase
intron 1 2
transcription
factors

62. Transcription
mature mRNA AAAAAAAAAA
RNA splicing
primary
transcript
transcription
factors RNA
exon 1 2 3 polymerase
intron 1 2
promotor

63. 3 types of RNA
 mRNA (messenger RNA)
 rRNA (ribosomal RNA)
 tRNA (transfer RNA)

64. Messenger RNA
 Delivers ______________ to the site of
Translation.

65.  mRNA instructions are written in
“3-nucleotide” sequences.
 These sequences are called codons.
 Ex.
 UUU, CUG, ACU, etc.
 There are 64 possible codons.

66. Translation
 Remember what happens in Transcription?
 DNA to RNA
 In Translation…RNA is coded for Amino
Acids.

67. Translation
 mRNA leaves the nucleus
 In the cytoplasm, ribosomes attach to the
mRNA ensuring the correct amino acid, for
each codon, is added to a growing chain of
amino acids which forms the resulting
protein.

68. Translation
polyadenylation site
(AATAAA)
cap structure
AAAAAAAAA 3’
mRNA
molecule
5’ Ribosome
cytoplasm

69. Translation
Peptide
chain
AAAAAAAAA 3’
mRNA
5’ molecule
Ribosome
cytoplasm

70.  Translation takes place
in the
______________
 tRNA (Transfer RNA)
molecules carry single
amino acids.
 They also have an
OPPOSITE “3-
nucleotide” sequence
called anticodons.

71.  rRNA (Ribosomal RNA) molecules are like
assembly lines  they carry:
 1 mRNA
 2 tRNA

73. 7 Steps in Translation
1 – mRNA start codon starts the process at the P
site.
2 – the next tRNA bonds to the next codon at the
A site.
3 – A & P are holding 2 tRNA’s…a
______________ bond is formed between 2
amino acids.

74. 4 – tRNA detaches from P-site, leaves behind
amino acid, leaves Ribosome.
5 – tRNA at A-site moves to the P-site. Now a
new codon is ready at the A-site for another
tRNA.

75. 6 – tRNA detaches from P-site, leaves behind
amino acid, ______________ ribosome.
7 – (Steps 2 – 6 repeat until a stop codon is
reached). Ex. UAG, UAA, UGA.
 A new protein is then released into the cell.

76. Translation

77. Translation

79. The Human Genome
 Only ~5% of our DNA actually codes for
proteins. Little variation exists from person to
person.The remainder is ‘junk’
 ‘Junk’ DNA includes repetitive sequences such
as micro and minisatellites. Varies a lot between
individuals allowing ‘DNA fingerprinting’

80. RNA polymerase responsible: transcription, and ribosome (which is
responsible for translation) are very accurate enzymes and make very few
mistakes.
IF RNA polymerase or a ribosome make a mistake it is not highly
detrimental to the cell

81.  An alteration in the nucleotide sequence of the
gene is called a ________________________ .
 Mutations in the gene affect the structure and
the function of all the proteins expressed from
the mutant gene.

82. Mutations
A change in the DNA sequence of the
gene
o Germline mutation (inherited)–
present in every cell in the body
o Somatic mutation (acquired) – present
only in the descendants of that cell
All cells acquire mutations as they
divide
Mutations can alter protein product
of DNA, stop gene working or
activate gene

83. Types of Mutation
(in coding sequence)
AGC TTC GAC CCG Wild type
AGC TGA CCC G Deletion
AGC TTC CCG ACC CG Insertion
AGC TTC TTC TTC GAC CCG Expansion
ATC TGC GAC CCG Point mutation
ATC TGA Nonsense ‘stop’

84. DNA Repair
• Cells require mechanisms to ___________________ DNA
damage
• Mutation rate reflects the number of damaging events in
DNA vs. the number of corrections
• 2 broad types of changes:
Single base mutations
Structural distortions

85. Repair Mechanisms
• Direct repair, e.g., photoreactivation of thymine dimers by a light-dependent enzyme
(photolyase)
• Nucleotide excision repair
• Mechanism
• Multiple systems, some of which act generally, others more specifically e.g. glycosylases
and AP endonucleases
• Found in bacteria, archaea and eucaryotes
• UV damage and repair of bulky lesions
• Tolerance systems allow replication of damaged DNA but with higher error frequencies
• Recombination repair uses homologous recombination to obtain the correct sequence from
an undamaged source
• Mismatch repair removes mismatches which arise during DNA replication

86. Nucleotide Excision Repair
Base mutation/distortion
5’ and 3’ incisions
Exonuclease excision
Replacement strand synthesis
Ligase seals backbone nick

87. Lectures 1-4 Overview
• DNA replication is semi-conservative: both
strands are used as templates for new strands
• DNA synthesis occurs exclusively 5’→3’
• DNA synthesis is semi-discontinuous (except in
rolling circle replication where the two strands
are replicated from independent origins):
Lagging strand synthesis
5’
3’ 3’
5’ 5’
3’
Leading strand synthesis
• Leading strand synthesis is primed once with a
primase-synthesized RNA primer
• Lagging strand synthesis is primed repeatedly
with Okazaki fragments

88. Summary
• DNA damage can be of a variety of types
• Cells require mechanisms to repair
DNA damage
• Different mechanisms exist
• Nucleotide excision repair is a sequential and coordinated
series of enzymatic events by which DNA damage cane be
repaired

89. Lectures 1-4 Overview
• DNA polymerases have proof-reading activity which
corrects incorporation of incorrect nucleotides
• Proteins at the replication fork:
DNA PolIII
Primase
Lagging strand
Helicase
(Okazaki fragment)
Parental DNA SSB
5’
3’ 3’
5’ 5’
3’
Leading strand + DNA PolI
+ Ligase
In eukaryotes replication is initiated at multiple ori
on different chromosomes; termination is poorly
understood

90. Exercise 1.
Each column in the table below represents three nucleotides. Within each column, fill in the cells that are
blank by using information from the cell that is not blank.
DNA strand TAC GGG
mRNA UCG CCU
Amino Acid Leu

91. Transcription and Translation problems
1. polypeptide: proline-tyrosine-histidine-valine-glutamic acid
What is the base sequence of the mRNA that codes for the polypeptide?
A. CCG-UAU-CAU-GUA-GAA
B. GAA-GUA-CAU-UAA-CCG
C. CCG-GUA-GAA-UAA-AUG
D. CCG-GUA-CAU-CUA-CCG
2. Use a genetic code table to determine the polypeptide sequence synthesized from the
mRNA below. Assume that translation begins at the first nucleotide at the 5' end.
5' AUGAAGUGUUAACCC 3‘
3. What RNA sequence is transcribed by the gene with the DNA template strand
5’ TTGAGCGCGTA 3’

92. 1. Major features of the Watson and Crick model of the double
helix
2. How DNA is replicated in Eukaryotes and Prokaryotes
3. The major steps involved in DNA replication
4. How the process of DNA replication ensures accuracy
5. Mutations and how they can be harmful or beneficial
6. Scientists Watson Crick, Meselson and Stahl, and Chargaff
discoveries
7. Chromosome structure and function
8. The purpose of Transcription and the differences between
RNA and DNA
9. How amino acids are designated in a stretch of RNA
10. Steps involved in protein synthesis, their locations, and the
important players involved
11. Be able to transcribe and translate DNA sequence