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Chapter 12
Molecular Biology
Of the Gene
• One gene differs from another only by the sequence of the nucleotide bases in DNA
• How does base sequence determine the uniqueness of a species or of individual traits between members of a species?
• DNA specifies proteins which make unique structures that make up all the characteristics of an organism
• DNA’s sequence of nucleotides sequence of amino acids specific enzymes structures
• In 1900’s, scientists knew the genetic material:• Must store information about development,
structure, and metabolic activities of a cell• Must be stable, so it could be replicated in cell
division and passed on • Must be able to undergo rare changes called
mutations to provide variability required for evolution
• In the1920’s, Frederick Griffith was working on a vaccine against Streptococcus pneumoniae
• Notices some colonies were shiny and smooth• Other colonies had a rough appearance
• Smooth colony individuals had a capsule, but those of the rough colony did not
• S strain caused death when injected into mice• R strain did not• Heat-killed S strain mixed with living R strain
caused death in mice when injected and when they were isolated from the mice had capsules
Fig. 12.1
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
• Oswald Avery worked on whether genetic material was protein or DNA
• Subjected materials to proteinases and a capsule was still produced
• Subjected materials to DNase and it was not• DNA was shown to be the genetic material• DNA structure:• Contains four nitrogenous bases • -Two purines- adenine (A) and guanine (G) that
were double ringed• -Two pyrimidines- thymine (T) and cytosine (C)
that were single ringed
• Percentage of each type of nucleotide differs from species to species
• Within a species, DNA has a constancy of bases• % of A always equals % of T and % of G always
equally % of C. These relationships are called Chargaff’s rules
Fig. 12.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
O
N
N
CH
CH
C
C
NH2
cytosine(C)
3C C2
C1
OHO P O
O
H
HH
HH
OH
CH3
O
HN
N
C
CH
C
C
OHO P O
O
H
HH
HH
OH
HN
N
N
CCH
O
C
CC
NH2N
C2
C2
C1
C1
OHO P O
O
guanine(G)
phosphate
H
HH
HH
OH
N
N
N
HCCH
NH2
C
CC
N
4
3C
2
C1
5 O
O
O
O
O
O
H
HH
HH
OH
c. Chargaff’s data
DNA Composition in Various Species (%)
Species
Homo sapiens (human)
Drosophila melanogaster (fruit fly)
Zea mays (corn)
Neurospora crassa (fungus)
Escherichia coli (bacterium)
Bacillus subtilis (bacterium)
31.0
27.3
25.6
23.0
24.6
28.4
31.5
27.6
25.3
23.3
24.3
29.0
19.1
22.5
24.5
27.1
25.5
21.0
18.4
22.5
24.6
26.6
25.6
21.6
A T G C
a. Purine nucleotides b. Pyrimidine nucleotides
nitrogen-containingbase
sugar = deoxyribose
thymine(T)
adenine(A)
HO P O CH2
5 CH2
5 CH2
5 CH2
C
4C
4C
4C
C
3C
3C
• Each human chromosome usually contains about 140 million base pairs
• Because any of the four nucleotides can be present at each position, the total possible nucleotide sequences is 4 to the 140th x 10 to the 6th or 4 to the 140,000,000th
• Rosalind Franklin’s work with x-ray diffraction determined that DNA was a double helix
Fig. 12.4
X-ray beam
b. c.
Rosalind Franklin
diffraction pattern
CrystallineDNA
diffractedX-raysa.
© Photo Researchers, Inc.; 12.4c: © Science Source/Photo Researchers, Inc.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
• Watson and Crick constructed a model of DNA and received the 1962 Nobel prize
• Polymers of nucleotides in a double helix form• Sugar-phosphate back bones on outside and
paired bases inside• Two DNA strands are antiparallel, meaning that
the sugar-phosphate groups of each strand are oriented in opposite directions
• 5’ end of one strand is paired to the 3’ end of the other strand
• Complementary base pairing means a purine always bonds to a pyrimidine
• They have an antiparallel arrangement to insure that bases are oriented properly so can interact
• This is the only model having molecular width revealed by Franklin’s x-ray diffraction pattern
Fig. 12.5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
P
P
P
P
c.
b.
Complementarybase pairing
sugar-phosphatebackbone
3.4 nm
2 nm
0.34 nm
P
P
S S4
5 end3 end
11
23
2 35
4
5
CG
G
C
C
G
T
T
A
A
C
Ga.
d.d.
a: © Kenneth Eward/Photo Researchers, Inc.; d: © A. Barrington Brown/Photo Researchers, Inc.
• Term DNA replication refers to process of copying a DNA molecule
• A template is a mold used to produce a shape complementary to itself
• During DNA replication, each DNA strand serves as a template for a new strand in a daughter molecule
• DNA replication is semiconservative replication because each daughter DNA double helix contains an old strand from parential DNA double helix and a new strand
Fig. 12.6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
region of parentalDNA double helix
region ofreplication:new nucleotidesare pairingwith those ofparental strands
region ofcompletedreplication
oldstrand
newstrand
daughter DNA double helix
daughter DNA double helix
A
A
A
A
A
A
A
A
AA
A
A
AA
A
A
A
A
A
A
T
T
T
T
T
TT
T
T
T
TT
T
G
G
G
G
G
G
GG
G
G
G
G
GG
G
G
C
C
C
CC
C
C
C
C
C C
C
C
oldstrand
newstrand
• Steps in replication:• 1) Unwinding of parental DNA is caused by the
breaking of weak hydrogen bonds between paired bases
• Enzyme called helicase necessary to unwind the molecule
• 2) Complementary base pairing occurs when new complementary nucleotides, always in the nucleus, are paired
• 3) Joining finishes replication by joining the complementary nucleotides to form new strands
• Each new daughter DNA molecule contains an old strand and a new strand
• Steps 2 and 3 are done with the enzyme complex called DNA polymerase
• DNA replication must occur before a cell can divide
• Cancer is characterized by rapid cell division• Sometimes treated with chemotherapeutic drugs
that are analogs to one of four nucleotides• Analogs have similar but not identical structure• They cause replication to stop and cells to die• Bacteria have a single circular loop of DNA that
must be replicated before the cell divides• Process begins at origin of replication site
• The strands are separated, unwound, and DNA polymerase binds to each side and begins copying
• The two DNA polymerases meet at a termination region, then the chromosomes separate
• Bacteria cells require about 40 minutes to replicate, but bacterial cells can divide every 20 minutes
• So replication can begin even before previous round is complete
• In eukaryotes, DNA replication begins at numerous origins of replication along the length of the chromosome
• Replication fork is the V shape of strands formed when replication bubbles spread bidirectionly until they meet
• Chromosomes are long and linear and replicate at about 500-5,000 base pairs per minute
• Because there are many individual origins of replication, the diploid DNA in humans of over 6 billion base pairs takes some hours
• In linear chromosomes, DNA polymerase cannot replicate to the ends
• Ends of chromosomes composed of telomeres which are short DNA sequences that are repeated over and over
• Telomeres are added back by the enzyme telomerase
• Eventually telomeres are lost and the cell cannot replicate
• In stem cells, this process preserves the ends of chromosomes and prevents the loss of DNA after successive rounds of replication
• DNA polymerase is very accurate and makes a mistake approximately once per 100,000 base pairs, but has proof reading capability so overall error rate is one in 100 million base pairs
Fig. 12.7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
replication fork replication bubble
parental strand
daughter strand
new DNAduplexes
a. Replication in prokaryotes
b. Replication in eukaryotes
replication isoccurring in
two directions
replication iscomplete
origin
• RNA is a polymer composed of nucleotides• Contains sugar ribose and bases adenine (A)
cytosine, (C), guanine (G),and uracil (U) which replaces the thymine in DNA
• Single stranded and does not form a helix• RNA comes in three major classes:• Messenger RNA (mRNA) takes message from
DNA in nucleus to ribosomes in cytoplasm• Transfer RNA (tRNA) transfers amino acids to
the ribosomes• Ribosomal RNA (rRNA) along with ribosomal
proteins, make up the ribosomes where polypeptides are synthesized
• Two major steps in synthesizing a protein from information in the DNA
• Transcription where one of the DNA strands acts as a template to make messenger RNA
• Translation where the messenger RNA directs the sequence of amino acids into a polypeptide
• Sequence of nucleotides in DNA to mRNA specify the order of amino acids in polypeptide
• Genetic code:• Codon is a triplet code where three nucleotides
code for one of the twenty amino acids• Code is degenerate meaning most amino acids
have more than one codon
• Code is unambiguous meaning each triplet codon has only one meaning
• Code has one start signal (AUG) and three stop signals (UAA) (UGA) and (UAG)
• Code is universal
Fig. 12.10
U C A G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
Second Base ThirdBase
FirstBase
CGAarginine
CGGarginine
AGUserine
AGCserine
AGAarginine
AGGarginine
GGUglycine
GGCglycine
GGAglycine
GGGglycine
UGGtryptophan
CGUarginine
CGCarginine
UGAstop
AUG (start)methionine
CAChistidine
CAAglutamine
CAGglutamine
AAUasparagine
AACasparagine
AAAlysine
AAGlysine
GAUaspartate
GACaspartate
GAAglutamate
GAGglutamate
UUUphenylalanine
CUUleucine
CUCleucine
CUAleucine
CUGleucine
AUUisoleucine
AUCisoleucine
AUAisoleucine
GUUvaline
GUCvaline
GUAvaline
GUGvaline
UCAserine
CCUproline
CCCproline
CCAproline
CCGproline
ACUthreonine
ACCthreonine
ACAthreonine
ACGthreonine
GCUalanine
GCCalanine
GCAalanine
GCGalanine
CAUhistidine
UAAstop
UCUserine
UAUtyrosine
UGUcysteine
UUCphenylalanine
UCCserine
UACtyrosine
UGUcysteine
UUAleucine
UCGserine
UAGstop
UUGleucine
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
• Messenger RNA has a sequence of bases complementary to a portion of one DNA strand
• When a gene is transcribed, a segment of DNA helix unwinds and unzips
• Rna nucleotides pair with complementary DNA nucleotides; this is known as the template strand
• RNA polymerase joins the nucleotides together in the 5’ 3’ direction or adds a nucleotide to the 3’ end of polymer under construction
• Transcription begins when RNA polymerase attaches to promoter in DNA
• Promoter defines the start of transcription, the direction of transcription, and the strand to be transcribed
• Initiation of transcription is the binding of RNA polymerase to promoter
• Elongation of mRNA continues until RNA polymerase comes to DNA stop codon sequence
• Causes release of mRNA now called mRNA transcript
• RNA transcript is called pre-mRNA and is modified before leaving the nucleus
• Receives a cap at 5’ end and a tail at 3’ end
• Cap is modified guanine (G) nucleotide which tells ribosome where to attach when translation begins
• Tail consists of 150-200 adenine (A) nucleotides• This poly-A tail helps transport of mRNA out of
nucleus and inhibits degration of mRNA by hydrolytic enzymes
• Pre-mRNA is composed of exons and introns• Exons are segments that will be expressed• Introns are segments in between exons that will
not be expressed• During pre-mRNA splicing, introns are removed
• In prokaryotes, introns splice themselves out• In eukaryotes, DNA splicing is done by
spliceosomes which contain small nuclear RNA (snRNA)
• Spliceosomes use ribozymes whose catalytic activity works like enzymes that are made of protein
• Presence of introns allows a cell to pick and chose which exons will go into a particular mRNA
• What is an exon in one mRNA could be an intron in another mRNA
• Called alternate mRNA splicing
• Some introns give rise to micro RNA (miRNA) which are involved in regulating the translation of mRNA
• Introns may also encourage crossing-over during meiosis, and permit exon shuffling which can play a role in evolution of new genes
Fig. 12.13
exon
intron intron
exon exonDNA
transcription
exon
intron intron
exon exon
5 3
exon
intron intron
exon exon
exon
intron RNA
exon
pre-mRNAsplicing
exon
cap poly-A tail
spliceosome
nucleus
mRNA
cytoplasm
nuclear porein nuclear envelope
cap poly-A tail
cap poly-A tail
3
3
5
5
pre-mRNA
3 5
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• Translation takes place in the cytoplasm of eukaryotic cells
• Transfer RNA (tRNA) molecules transfer amino acids to the ribosomes
• tRNA is a single stranded nucleic acid that doubles back on itself to create regions where complementary bases hydrogen bond to one another
• There is at least one tRNA molecule for each of the 20 amino acids
• Amino acids bind to 3’ end of tRNA• The opposite end contains an anticodon that is
complementary to a specific mRNA codon
Fig. 12.14
G
GA
A A
CCC CC CU U UU
Hydrogenbonding
codon
anticodon
mRNA35
aminoacid
leucine
3
5
anticodon end
amino acid end
b.a.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
• In eukaryotes, ribosomal RNA (rRNA) is produced from a DNA template in the nucleolus of a nucleus
• Packaged with proteins into two ribosomal subunits one larger than the other
• They move into the cytoplasm where they combine when translation begins
• May remain in cytoplasm or attach to endoplasmic reticulum
• Ribosomes have a binding site for mRNA and three binding sites for tRNA
• When ribosome moves down a mRNA molecule, the polypeptide increases by one AA at a time
• Several ribosomes are often attached to and translating the same mRNA
• The entire complex is called a polyribosome
Fig. 12.15
tRNA bindingsites
outgoingtRNA
3
mRNA
incomingtRNA
polypeptide
5
small subunit
mRNA
large subunit
a. Structure of a ribosome b. Binding sites of ribosome
c. Function of ribosomes d. Polyribosome
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Courtesy Alexander Rich
• Translation requires three steps:• 1) Initiation brings all the translation components
together• Initiation factors (proteins) assemble small
ribosome subunit, mRNA, initiator tRNA, and large ribosomal subunit
• 2) Elongation where polypeptide increases in length one amino acid at a time
• Requires elongation factors (proteins) for binding tRNA anticodons to mRNA codons
• 3) Termination of polypeptide occurs at a stop codon
• Requires protein called release factor which binds to stop codon and cleaves the polypeptide from the last tRNA
Fig. 12.17
UA
C AUG
C U G
G A C
UA
C AUG G A C
C U G
Elongation
A tRNA–amino acidapproaches theribosome and bindsat the A site.
Two tRNAs can be at aribosome at one time;the anticodons arepaired to the codons.
anticodon
tRNApeptidebond
asp
35 5
Asp
1 2 4
UA
C AUG G A C
C U G
UC
A
G A C
C U G
AUG
U G G
A C C
Peptide bond formationattaches the peptidechain to the newlyarrived amino acid.
The ribosome moves forward; the“empty” tRNA exits from the E site;the next amino acid–tRNA complexis approaching the ribosome.
35
Met
Val
Asp
Ala
Trp
Ser
3 35
Met
Val
Asp
Ala
Trp
Ser Thr
3
peptidebond
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Met
Ala
Trp
Ser
Val
Met
Ala
Trp
Ser
Val
Fig. 12.18
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
The ribosome comes to a stopcodon on the mRNA. A releasefactor binds to the site.
The release factor hydrolyzes the bondbetween the last tRNA at the P site andthe polypeptide, releasing them. Theribosomal subunits dissociate.
3
release factorAla
Trp
Val
Asp
Glu
3
5
UA
UA U G A
AG A
U G A
UC
U
Termination
stop codon
• Gene has been expressed once its product is made and is operating in the cell
• Eukaryotic chromosome contains a single double helix DNA molecule, but is composed of more than 50% protein
• Histones are the large majority and play a primary structural role
• A human cell contains at least 2 meters of DNA