From Gene to Protein
Lecture 14
Fall 2008
Function of DNA
• Genotype
– Sequence of nucleotide bases in DNA
– All the alleles of every gene present in a given
individual
• Gene : a discrete unit of hereditary information consisting of a
specific nucleotide sequence in DNA
• Phenotype
– Any observable traits in an individual
• Physical, physiological & behavioral
1
Function of DNA
2
How does the genotype
produce the phenotype?
• Gene expression
• Creates proteins from DNA
– “one gene – one enzyme”
– “one gene – one protein”
– The function of a gene is to
dictate the production of a
polypeptide
Fig.17.3
3
Function of DNA
Two processes:
• Transcription
– The transfer of genetic
information from DNA into
RNA
– Synthesis of RNA
– Nucleus
• Translation
– The transfer of information
from RNA into proteins (via the
ribosomes)
– Synthesis of polypeptide
– Cytoplasm
Fig. 17.3
Protein Structure
Primary structure
• Polypeptide chain
– Unique sequence of amino
acids
• Amino acids
– 20 types
– Basic structure of each
amino acid is the same
– Unique side group = R
group
See Fig. 5.17
4
5
Overview of Transcription & Translation
Transcription
• “rewrites” DNA into
RNA
• Still in nucleic acid
“language”
Translation
• Converts nucleic acid
language into
polypeptide (amino
acid) language
Fig. 17.4
6
Overview of Transcription & Translation
• Triplet code of DNA
– Non-overlapping
sequence of 3 bases
that code for amino
acid
• 64 possible
combinations (43)
• RNA complementary
to DNA
Fig. 17.4
The Dictionary of the Genetic Code
7
Codons
• mRNA triplets
• 64 possible
combinations (43)
–
–
–
–
Only 20 amino acids
Redundancy
Start code
Stop code
Fig. 17.5
Transcription
Transcription
• The transfer of genetic
information from DNA
into RNA
3 main steps
• Initiation of Transcription
• RNA Elongation
• Termination of
Transcription
8
Transcription
Initiation
• DNA strand separates at promoter
– Specific area of DNA that designates the start of a gene
– Contains start point and several dozen nucleotide pairs
– Determines template strand
• RNA polymerase binds at promoter
– Unwinds DNA strand & joins nucleotides
• RNA synthesis begins
– Nucleotides that will form RNA line up with DNA nucleotides
• U replaces T in the RNA strand
• Bases of RNA & DNA joined by hydrogen bonds
– Doesn’t need a primer
– 5’to 3’
Fig. 17.7
9
Transcription
• Transcription Factors
– Mediate binding of
RNA polymerase to
promoter & initiation
(eukaryotes)
– One TF binds to
TATA box
• Transcription initiation
complex
– Complex of RNA
polymerase and
transcription factors
Fig. 17.8
10
Transcription
11
RNA Elongation
• RNA synthesis
continues
– ~10-20 DNA bases
exposed at a time
– ~ 40
nucletides/second
(eukaryotes)
• “peels off” of DNA
• DNA strands come
back together after
RNA removed
Fig. 17.7
Transcription
Termination of Transcription
• Bacteria
– RNA polymerase reaches terminator sequence
• Signals the end of the gene
• RNA polymerase detaches from DNA & RNA
• Eukaryotes
– Polyadenylation signal sequence
– DNA segment (UAAUAAA)
– RNA cut ~10-35 nucleotides downstream of signal
• RNA polymerase continues transcribing until stopped by
enzyme
Fig. 17.7
12
RNA Processing
13
Primary transcript (pre-mRNA) altered
• Addition of 5” cap and poly-A tail
• 5’ cap
– Modified guanine nucleotide
– Occurs soon after RNA synthesis begins
• Poly-A tail
– 50-250 adenine nucleotides
• Function
– Facilitate export from nucleus
– Protection from degradation by hydrolytic enzymes
– Facilitate attachment of ribosome to 5’ end
• Untranslated regions (UTR)
Fig. 17.9
14
RNA Processing
• RNA splicing
– Removal of introns (intervening sequences)
• Intron: non-coding regions of nucleotides
– Joining of exons (expressed sequences)
• Exons : coding regions of nucleotides
– Exceptions – UTRs (untranslated regions)
• Average polypeptide = 400 (1200 base pairs)
• Average transcription unit = 27,000 base pairs
• Results in mRNA (messenger RNA) which then
leaves the nucleus
Fig. 17.10
RNA Processing
• Spliceosome
• Complex of small nuclear
ribonucleoproteins
(snRNPs) and other
proteins
• Binds w/pre-RNA
– Intron carries region
recognized by snRNPs
(snRNA)
– Cuts out introns
– Binds exons together
Fig. 17.11
15
16
RNA Processing
• Ribozymes
– RNA molecules that function as enzymes
• RNA splicing
– In some organisms
• Three characteristics
– Single strand allows RNA to base pair with itself to
form particular 3-D structure
– Some functional groups on bases can act in catalysis
– Ability to hydrogen bond with other nucleic acids adds
specificity of catalytic activity
17
Translation
Translation
• Conversion of nucleic acid language into
polypeptide (amino acid) language
• Synthesis of a polypeptide
3 main steps
• Initiation
• Elongation
• Termination
Translation
18
Transfer RNA (tRNA)
• Converts the codon of mRNA
into an amino acid
• Anticodon at one end
–
•
Complementary pairing with codon
on mRNA
Amino acid attachment site at
other end
Fig. 17.14
Translation
19
• Attachment of amino acid
to tRNA
– ATP
– Aminoacyl-tRNA
synthetases
• 20 types
• More codons than tRNAs
– ~45 different tRNAs
– “wobble”
• Some tRNAs able to bind to
more than one codon
• 3rd base has flexibility
Fig. 17.15
20
Translation
• Ribosomes are site of
translation
– Made of proteins &
ribosomal RNA (rRNA)
– Small & large subunits
• Made in nucleolus
• Exported to cytoplasm
– Aminoacyl-tRNA binding
site
– Peptidyl-tRNA binding site
– Exit site
Fig. 17.16
21
Translation
Initiation
Translation initiation complex formed
• mRNA binds to small subunit
• Initiator tRNA (UAC anticodon) binds to mRNA at start
codon (AUG)
• Large ribosomal subunit binds to small subunit
– tRNA positioned in P site
• Requires initiation factors & GTP
Fig. 17.17
Translation
22
Elongation
• Requires elongation
factors & GTP
• Codon recognition
• Peptide bond formation
– Catalzed by rRNA part of
large ribosomal subunit
• Translocation
Fig. 17.18
Translation
23
Termination
• When stop codon on mRNA reaches the A site
– UAG, UAA, UGA
– Release factors bind to stop codon at A site
• Adds H2O molecule to break bond between polypeptide and tRNA in P
site
• Polypeptide chain free
Fig. 17.19
Translation
• Polyribosomes
– Multiple
ribosomes
translating same
strand of mRNA
simultaneously
– Increases speed
of translation
Fig. 17.20
24
From Genotype to Phenotype
Fig. 17.25
25
26
Protein Completion
• Poypeptide chain folds spontaneously as it is
being synthesized
– Chaparonins
• Post-translational modifications
– Additions of sugars, lipids, phosphate groups
– Removal of amino acids from end of polypeptide
chain
– Quaternary structure formed
27
Targeting Proteins
• Free & bound ribosomes
• Polypeptide synthesis always begins in cytosol
• Signal peptide
– Sequence of ~ 20 amino acids at leading end of polypeptide
– Targets protein for ER
• Signal-recognition particle (SRP)
– Brings ribosome to translocation complex
• Protein complex in ER with recognition proteins – forms pore
Fig. 17.21
28
Mutations
Mutation
• Change in the nucleotide sequence of DNA
– May give rise to an altered protein
• Point mutations
– Chemical changes in a single base pair of a
gene
Fig. 17.22
29
Types of Point Mutations
• Base-pair
substitutions
– Replacement of one
nucleotide and its
partner with another
nucleotide pair
– Silent mutation
• No effect on
polypeptide structure
• Redundancy in codons
Types of Point Mutations
• Missense mutation
– Changes one amino acid to another
• May not affect function of protein
• May significantly alter protein function
• Nonsense mutations
– Changes amino acid codon to a stop codon
– Terminates translation early
• Nonfunctional protein
30
Types of Point Mutations
Insertions or deletions
• Frameshift mutation
– Alters reading frame
Fig. 17.23
31
Mutations
• Can be spontaneous
– Errors during DNA replication or recombination
• Rare: In DNA replication, only 1 in a billion bases incorrectly
paired
• Cell has some repair mechanisms
• Can be caused by mutagen
– Physical or chemical agent that causes change
• E.g., x-rays, UV light, agent orange, tobacco
• Spontaneous mutations are rare, but rate of
mutation increased by mutagens
32
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Mutations and Evolution
• If mutation occurs in somatic cells, the mutation
is not passed on to offspring
• If mutation occurs in cells that produce gametes,
mutation can be passed on to offspring
• Mutations are one way of introducing new alleles
– Increases genetic diversity in populations