2. History
• Archibald Garrod – 1909
• First to suggest that genes dictate
phenotype through production of
proteins
• Believed that genetic diseases resulted
from the inability to make particular
enzymes
• “Inborn errors of metabolism”
3. One Gene – One Enzyme
• Beadle & Ephrussi – 1930’s
• 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
– Wild type Neurospora grows on minimal medium (agar
enriched with a few nutrients)
– 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
– That identified the defective synthetic pathway
– 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 – 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
– Many copies of an RNA can be made from one
copy of DNA.
• Greater regulation of gene expression
– Specific controls can act at each step in the
pathway between DNA and proteins.
– 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
– consists of a sugar-phosphate backbone, with
nucleotides attached to the 1' C of the sugar.
• Differences between DNA and RNA :
– Contains the sugar ribose instead of
deoxyribose
– The nucleotide, uracil, is substituted for
thymine
– 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
– A copy of a gene.
– Has a sequence complementary to one strand of
the DNA & identical to the other strand.
– Carries the information stored in DNA in the
nucleus to the ribosomes in the cytoplasm where
protein is made.
• tRNA - transfer RNA
– A small RNA with a very specific structure that
can bind an amino acid at one end, and mRNA at
the other end.
– Acts as an ‘adaptor’ to carry & attach amino acids
to the appropriate place on the mRNA.
12. Types of RNA (Cont.)
• rRNA - ribosomal RNA
– One of the structural components of the
ribosome.
– Has a sequence complimentary to regions of
the mRNA
– Allows ribosome to bind to an mRNA
• snRNA - small nuclear RNA
– 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
– there are only 4 nucleotides and 20 amino acids.
– 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
– In many cases, codons that are synonyms differ only in
the third base of the triplet
• Different organisms have different frequencies of
codon usage.
– 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 “stop” (or “start)
• There is no ambiguity
– 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
– 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 3’ end
• So only one mRNA is made, elongating 5’ 3’
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
– 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
– 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 5’ to 3’ 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
– 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.
– The ribosome falls off the mRNA,
– That signals termination by the RNA polymerase.
– 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 5’ end is capped with modified guanine
– Protects mRNA from degradation
– Helps attach the ribosome
• At the 3’ end an enzyme makes a poly-A tail
formed from 50 to 250 adenine nucleotides
– Inhibits degradation and helps ribosome attach
– 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
– 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
– 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
– 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
– The A site accepts a new tRNA bearing an amino
acid
– the P site bears the tRNA attached to the growing
chain.
– 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;
– This protein can only bind to that particular
tRNA and attach the correct amino acid to the
acceptor site.
– 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
– 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
– 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
– 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
– Codes for methionine.
– Not every protein starts with methionine,
– 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 ribosome’s 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.
– For example, a tyrosine residue might be phosphorylated.
• Become glycosylated.
– 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:
– Chromosomal mutations
– Point mutations
– 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:
– Translocations
– Inversions
– Deletions
– Nondisjunction
62. Translocations & Inversions
• Translocation
– The interchange of large segments of DNA
between two chromosomes.
– Can change gene expression if a gene is at the
translocation breakpoint or if it is reattached so that
it is incorrectly regulated
• Inversion
– Occurs when a region of DNA flips its orientation
with respect to the rest of the chromosome.
– Rotates, end for end
– This can lead to the same problems as
translocations.
63. Deletions & Nondisjunction
• Deletion
– Sometimes large regions of a chromosome are deleted.
– This can lead to a loss of important genes.
• Nondisjunction
– Sometimes chromosomes do not divide correctly in cell
division
– When large regions of a chromosome are altered (such as
translocation), the chromosome may not segregate properly
during cell division
– One daughter cell will end up with extra genetic material,
one will end up with less than its share
– This is called nondisjunction.
– 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
– Creates a stop codon where none previously existed.
– This shortens the resulting protein, possibly removing
essential regions.
• Missense mutation
– Changes the code of the mRNA.
– Which changes the resulting amino acid
– This may alter the shape and properties of the protein.
• Silent mutation
– Has no effect on protein sequence.
– Because the genetic code is redundant, some changes
have no effect
65.
66. Frameshift Mutations
• Insertions or deletions have a disastrous
effect
• mRNA is “read” 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