2. History
⢠Archibald Garrod â 1909
⢠First to suggest that genes dictate
phenotype through production of
proteins
⢠Believed that genetic diseases resulted
from the inability to make particular
enzymes
⢠âInborn errors of metabolismâ
3. One Gene â One Enzyme
⢠Beadle & Ephrussi â 1930âs
⢠Studied mutations affecting eye color in
Drosophila
⢠Concluded that each mutation blocks
pigment synthesis at a specific step by
preventing production of the enzyme that
catalyzes that step
⢠Specific pathways were not known, so
results were inconclusive
4. Beadle & Tatum
⢠Treated Neurospora (a mold) with X-rays
⢠Looked for mutations in nutritional requirements
â Wild type Neurospora grows on minimal medium (agar
enriched with a few nutrients)
â All mutants will grow on complete medium (agar plus all 20
amino acids & other nutrients)
⢠Identified the specific amino acid required for growth
by each mutant
â That identified the defective synthetic pathway
â Looked at each intermediate step in the blocked synthetic
pathway
⢠Concluded that mutation in a single gene blocked
production of a single enzyme
5.
6. One Gene â One Polypeptide
⢠Not all proteins are enzymes
⢠Can extend one gene = one enzyme
doctrine to one gene = one polypeptide
⢠Many proteins are comprised of two or
more polypeptides
7. Central Dogma
⢠How does the sequence of a strand of DNA
correspond to the amino acid sequence of a
protein?
⢠The central dogma of molecular biology, states
that:
8. Transcription & Translation
⢠DNA is first copied (transcribed) to an
RNA intermediate
⢠The RNA intermediate is then translated
to protein
⢠Why have an intermediate between
DNA and the proteins it encodes?
9. Why RNA?
⢠The DNA remains protected in the nucleus,
away from caustic enzymes in the
cytoplasm.
⢠Gene information can be amplified
â Many copies of an RNA can be made from one
copy of DNA.
⢠Greater regulation of gene expression
â Specific controls can act at each step in the
pathway between DNA and proteins.
â The more elements there are in the pathway, the
more opportunities there are for control
10. What is RNA?
⢠RNA has the same primary structure as DNA
â consists of a sugar-phosphate backbone, with
nucleotides attached to the 1' C of the sugar.
⢠Differences between DNA and RNA :
â Contains the sugar ribose instead of
deoxyribose
â The nucleotide, uracil, is substituted for
thymine
â RNA exists as a single-stranded molecule.
⢠Because of the extra hydroxyl group on the sugar,
RNA is too bulky to form a a stable double helix.
⢠Regions of double helix can form where there is
some base pair complementation resulting in hairpin
loops.
11. Types of RNA
⢠mRNA - messenger RNA
â A copy of a gene.
â Has a sequence complementary to one strand of
the DNA & identical to the other strand.
â Carries the information stored in DNA in the
nucleus to the ribosomes in the cytoplasm where
protein is made.
⢠tRNA - transfer RNA
â A small RNA with a very specific structure that
can bind an amino acid at one end, and mRNA at
the other end.
â Acts as an âadaptorâ to carry & attach amino acids
to the appropriate place on the mRNA.
12. Types of RNA (Cont.)
⢠rRNA - ribosomal RNA
â One of the structural components of the
ribosome.
â Has a sequence complimentary to regions of
the mRNA
â Allows ribosome to bind to an mRNA
⢠snRNA - small nuclear RNA
â Is involved in the machinery that processes
RNA's as they travel between the nucleus and
the cytoplasm.
13. The Genetic Code
⢠How does mRNA specify an amino acid
sequence?
⢠It would be impossible for each amino acid to
be specified by one nucleotide
â there are only 4 nucleotides and 20 amino acids.
â two nucleotide combinations could only specify 16
amino acids.
⢠Each amino acid is specified by a
combination of three nucleotides, called a
codon
14.
15.
16. The Code is Redundant, Not
Ambiguous
⢠Each amino acid may be specified by up to six
codons
â In many cases, codons that are synonyms differ only in
the third base of the triplet
⢠Different organisms have different frequencies of
codon usage.
â A giraffe might use CGC for arginine much more often
than CGA, and the reverse might be true for a sperm
whale.
⢠Some codons specify âstopâ (or âstart)
⢠There is no ambiguity
â the same codon ALWAYS codes for the same amino acid
17. Codons & Anticodons
⢠How do tRNAs recognize to which codon
to bring an amino acid?
⢠The tRNA has an anticodon on its mRNA-
binding end
⢠The anticodon is complementary to the
codon on the mRNA.
⢠Each tRNA only binds the appropriate
amino acid for its anticodon
19. Transcription
⢠How does the sequence information from
DNA get transferred to mRNA?
⢠How is this information carried to the
ribosomes in the cytoplasm?
⢠This process is called transcription
⢠Highly similar to DNA replication.
⢠Different enzymes are used in
transcription.
⢠The most important is RNA polymerase
20. RNA Polymerase
⢠RNA polymerase is a holoenzyme
â an agglomeration of many different factors
⢠Together, direct the synthesis of mRNA
⢠Pries the DNA strands apart
⢠Strings complimentary RNA nucleotides on the
DNA template
⢠Like DNA polymerase, can only add to the 3â end
⢠So only one mRNA is made, elongating 5â ď 3â
23. Initiation
⢠RNA polymerase must recognize the beginning of
a gene to know where to start synthesizing mRNA.
⢠One part of the enzyme has a high affinity for a
particular DNA sequence that appears at the
beginning of genes.
⢠The sequence where RNA polymerase attaches to
the DNA and begins transcription = the promoter
â a unidirectional sequence on one strand of the DNA
⢠Tells RNA polymerase both where to start and in
which direction (that is, on which strand) to
continue synthesis.
24. The Promoter
⢠In prokaryotes, RNA polymerase recognizes
and binds the promoter
⢠The bacterial promoter almost always
contains some version of the following
elements:
25. Eukaryotic Promoters
⢠In eukaryotes special proteins, transcription factors,
mediate binding RNA polymerase and the
promoter
⢠RNA polymerase binds to the promoter only after
transcription factors bind
⢠Transcription factors + RNA polymerase, bound to
the promoter = transcription initiation complex
⢠Eukaryotic promoters usually include a TATA box
â A nucleotide sequence containing TATA about
25 nucleotides prior to the start point
26.
27. Elongation
⢠The RNA polymerase stretches open the
double helix at the start point in the DNA
and begins synthesis of a complementary
RNA strand on one of the DNA strands
⢠The RNA polymerase recruits RNA
nucleotides in the same way that DNA
polymerase recruits dNTPs.
⢠Since synthesis only proceeds in the 5' to 3'
direction, there is no need for Okazaki
fragments.
28.
29. Sense & Antisense
⢠Synthesis only occurs in the 5â to 3â direction
⢠In transcription, only one DNA strand is
copied
⢠We call the strand that is copied the
antisense or template strand
⢠The other strand, which is identical to the
mRNA made (substituting U for T), is the
sense or coding strand.
30. Termination of Transcription
⢠How does RNA polymerase know when to stop
transcribing a gene?
⢠Sequence that signals the end of transcription =
terminator
⢠RNA polymerase transcribes the terminator
â The transcribed terminator actually ends the process
⢠In prokaryotes there is no nucleus, so ribosomes can
begin making protein from an mRNA immediately
⢠The terminator sequence of the mRNA allows it to
form a hairpin loop, which blocks the ribosome.
â The ribosome falls off the mRNA,
â That signals termination by the RNA polymerase.
â RNA polymerase falls off the DNA and transcription
31. Eukaryotic Termination
⢠RNA polymerase continues for hundreds of
nucleotides beyond the termination signal
⢠AAUAAA
⢠At a point 10 to 35 nucleotides past the
AAUAAA, the forming m-RNA is cut free
⢠The cleavage site is the point of addition of
a poly-A tail
32. Post Transcription
Modification
⢠In eukaryotes, enzymes modify pre-mRNA before
it is sent to the cytoplasm
⢠Both ends of the transcript are altered
⢠The 5â end is capped with modified guanine
â Protects mRNA from degradation
â Helps attach the ribosome
⢠At the 3â end an enzyme makes a poly-A tail
formed from 50 to 250 adenine nucleotides
â Inhibits degradation and helps ribosome attach
â May also help export mRNA out of the nucleus
⢠Interior sections are cut out, and the remaining
parts are spliced together
34. Introns & Exons
⢠Most eukaryotic genes and their RNA
transcripts have long noncoding stretches of
nucleotides = introns
⢠Noncoding sequences are interspersed
between coding sections
⢠Coding sections = exons
⢠That is, the sequence of eukaryotic DNA that
codes for a polypeptide is not continuous
⢠RNA polymerase transcribes both introns and
exons
35. RNA Splicing
⢠Introns are cut out and exons are spliced
together before mRNA exits the nucleus
⢠Short nucleotide sequences at the end of
introns are signals for RNA splicing
⢠Small nuclear ribonucleoproteins
(snRNPs) recognize splice sites
â Composed of snRNA & protein
⢠Several snRNPs and additional proteins
form a complex = spliceosome
⢠At splice sites at the end of an intron, cuts
out the intron and fuses the exons
37. Why Introns?
⢠Introns may play regulatory role in the cell
⢠Split genes allow a single gene to code more
than one kind of polypeptide
⢠Outcome depends on which sections are
treated as exons during RNA processing
â Alternative RNA splicing
⢠May facilitate evolution of new proteins
⢠Increase possibility of potentially beneficial
crossing-over of genes
38.
39. Translation
⢠How do messenger RNAs direct protein
synthesis?
⢠The message encoded in the mRNA is an
amino acid sequence
⢠mRNA travels to ribosome in the
cytoplasm, where the message is read
⢠The specified amino acids are assembled
on the mRNA template on the ribosome
⢠Enzymes help form the sequenced amino
acids into a polypeptide
40.
41. The Ribosome
⢠The cellular factory where proteins are synthesized
⢠Consists of structural RNA and ~ 80 different proteins.
⢠In its inactive state, it exists as two subunits
â a large subunit and a small subunit.
⢠When the small subunit encounters an mRNA, it
begins translation of the mRNA to protein.
⢠There are three sites in the large subunit
â The A site accepts a new tRNA bearing an amino
acid
â the P site bears the tRNA attached to the growing
chain.
â The E site contains the exiting tRNA
42.
43. Charging the tRNA
⢠tRNA (transfer RNA) acts as a translator between
mRNA and protein
⢠Each tRNA has a specific anticodon and an amino
acid acceptor site.
⢠Each tRNA also has a specific charger protein;
â This protein can only bind to that particular
tRNA and attach the correct amino acid to the
acceptor site.
â These charger proteins are called aminoacyl
tRNA synthetases
⢠The energy to make this bond comes from ATP.
44.
45. Aminoacyl-tRNA Synthases
⢠Each tRNA must match with the correct amino acid
â Each tRNA must attach only the amino acid specified by
the mRNA codon to which the tRNA anticodon binds
⢠The amino acid is joined to the tRNA by an
aminoacyl-tRNA synthase
â There are 20 of these enzymes; one for each amino acid
⢠Catalyzes the covalent bond between the amino acid
and tRNA
⢠The active site of each aminoacyl-tRNA synthase fits
only a specific amino acid and tRNA
⢠Once the amino acid is bound, the tRNA is
aminoacyl tRNA
46.
47. Wobble
⢠If there was one tRNA for each mRNA
codon, there would be 61 different tRNAs
⢠Actually, there are fewer
⢠Some tRNAs have anticodons that
recognize 2 or more different codons
⢠Base pairing rules between the third base of
a codon and its tRNA anticodon are not a
rigid as DNA to mRNA pairing
â Example: U in tRNA can pair with either A or T
in the third position of an mRNA codon
⢠This flexibility is called wobble
48.
49. Initiation of Translation
⢠The start signal for translation is the codon
ATG
â Codes for methionine.
â Not every protein starts with methionine,
â Often this first amino acid will be removed in
post-translational processing.
⢠A tRNA charged with methionine binds to the
translation start signal.
⢠The large subunit binds to the mRNA and the
small subunit
⢠Elongation begins.
50.
51. Elongation of the New Protein
⢠After the first charged tRNA appears in the A site, the
ribosome shifts so that the tRNA is in the P site.
⢠New charged tRNAs, corresponding the codons of
the mRNA, enter the A site, and a peptide bond is
formed between the two amino acids.
⢠The first tRNA is now released
⢠The ribosome shifts again so that a tRNA carrying
two amino acids is now in the P site
⢠A new charged tRNA can bind to the A site.
⢠This process of elongation continues until the
ribosome reaches a stop codon.
52.
53. Termination of the Protein
⢠When the ribosome reaches a stop
codon, no aminoacyl tRNA binds to the
empty A site.
⢠This is the ribosomeâs signal to break
into its large and small subunits,
⢠Releasing the new protein and the
mRNA.
54.
55. Polyribosomes
⢠A single mRNA can be used to make
many copies of a polypeptide at the
same time
⢠Multiple ribosomes can read the same
mRNA strand, like beads on a string
⢠These strings are called polyribosomes
57. Post-Translational Processing
⢠This isn't always the end of the story for the new
protein.
⢠Often it will undergo post-translational modifications.
⢠Modifications include:
⢠Cleavage by a proteolytic (protein-cutting) enzyme at
a specific place.
⢠Having some amino acids altered.
â For example, a tyrosine residue might be phosphorylated.
⢠Become glycosylated.
â Many proteins have carbohydrates covalently attached to
asparagine residues.
58.
59. Mutations
⢠What kinds of errors can occur in DNA?
⢠What causes them?
⢠What are their effects?
⢠Types of mutations:
â Chromosomal mutations
â Point mutations
â Frameshift mutations
60.
61. Chromosomal Mutations
⢠Mutations that occur at a macroscopic
level.
⢠Large sections of chromosomes can be
altered or shifted, leading to changes in
the way genes are expressed.
⢠Types of chromosomal mutations:
â Translocations
â Inversions
â Deletions
â Nondisjunction
62. Translocations & Inversions
⢠Translocation
â The interchange of large segments of DNA
between two chromosomes.
â Can change gene expression if a gene is at the
translocation breakpoint or if it is reattached so that
it is incorrectly regulated
⢠Inversion
â Occurs when a region of DNA flips its orientation
with respect to the rest of the chromosome.
â Rotates, end for end
â This can lead to the same problems as
translocations.
63. Deletions & Nondisjunction
⢠Deletion
â Sometimes large regions of a chromosome are deleted.
â This can lead to a loss of important genes.
⢠Nondisjunction
â Sometimes chromosomes do not divide correctly in cell
division
â When large regions of a chromosome are altered (such as
translocation), the chromosome may not segregate properly
during cell division
â One daughter cell will end up with extra genetic material,
one will end up with less than its share
â This is called nondisjunction.
â When there are extra or too few copies of a gene, the cell
will have problems
64. Point Mutations
⢠Point mutations are single base pair changes.
⢠Three possible outcomes:
⢠Nonsense mutation
â Creates a stop codon where none previously existed.
â This shortens the resulting protein, possibly removing
essential regions.
⢠Missense mutation
â Changes the code of the mRNA.
â Which changes the resulting amino acid
â This may alter the shape and properties of the protein.
⢠Silent mutation
â Has no effect on protein sequence.
â Because the genetic code is redundant, some changes
have no effect
65.
66. Frameshift Mutations
⢠Insertions or deletions have a disastrous
effect
⢠mRNA is âreadâ as a series of three letter
words
⢠Insertions or deletions that are not
multiples of three, shift the reading frame
67. Frameshift Example
⢠Given the coding sequence:
AGA UCG ACG UUA AGC
⢠corresponding to the protein:
arginine - serine - threonine - leucine - serine
⢠The insertion of a C-G base pair between bases 6
and 7 would result in the following new code:
AGA UCG CAC GUU AAG C
⢠which would result in a non-functional protein:
arginine - serine - histidine - valine - lysine
⢠Every amino acid after the insertion will be wrong.
⢠The frame shift might even generate a stop codon
which would prematurely end the protein.
68.
69. DNA Repair
⢠If replication of DNA proceeded as was described
previously, DNA polymerase would make a
mistake on average about every 1000 base pairs.
⢠This level would be unacceptable, because too
many genes would be rendered non-functional.
⢠Organisms have elaborate DNA proofreading and
repair mechanisms, which can recognize false
base-pairing and DNA damage, and repair it.
⢠The actual error rate is more in the region of one
in a million to one in a billion.
70. The Beauty of Mutations
⢠Why mutations?
⢠Our environment constantly changes, the Earth
and its ecosystems change.
⢠Populations must change to survive
⢠Evolutionary change requires variation, the raw
material on which natural selection works
⢠One mechanism for variation and change is at the
DNA level.
⢠Mutations can be beneficial and enable the
organism to adapt to a changing environment.
⢠However, most mutations are deleterious, and
cause varied genetic diseases