Chapter 17
From Gene to Protein
Question?
• How does DNA control a cell?
• By controlling Protein Synthesis.
• Proteins are the link between genotype and
phenotype.
For tests:
• Name(s) of experimenters
• Outline of the experiment
• Result of the experiment and its importance
1909 - Archibald Garrod
• Suggested genes control enzymes that
catalyze chemical processes in cells.
• Inherited Diseases - “inborn errors of
metabolism” where a person can’t make an
enzyme.
Example
• Alkaptonuria - where urine turns black after
exposure to air.
• Lacks - an enzyme to metabolize alkapton.
1941 - George Beadle and Edward Tatum
• Worked with Neurospora and proved the link
between genes and enzymes.
Neurospora
Pink bread mold
Experiment
• Grew Neurospora on agar.
• Varied the nutrients.
• Looked for mutants that failed to grow on
minimum agar.
Results
• Three classes of mutants for Arginine
Synthesis.
• Each mutant had a different block in the
Arginine Synthesis pathway.
Conclusion
• Mutations were abnormal genes.
• Each gene dictated the synthesis of one
enzyme.
• One Gene - One Enzyme Hypothesis.
Current Hypothesis
• One Gene - One Polypeptide Hypothesis
(because of quaternary (4th degree) structure).
Central Dogma
DNA
Transcription
RNA
Translation
Polypeptide
Explanation
• DNA - the Genetic code or genotype.
• RNA - the message or instructions.
• Polypeptide - the product for the phenotype.
Genetic Code
• Sequence of DNA bases that describe which
Amino Acid to place in what order in a
polypeptide.
• The genetic code gives the primary protein
structure.
Code Basis
If you use:
• 1 base = 1 amino acid
• 4 bases = 4 amino acids
• 41 = 4 combinations, which are not enough for
20 AAs.
If you use:
•
•
•
•
2 bases = 1 amino acid
Ex – AT, TA, CA, GC
42 = 16 amino acids
Still not enough combinations.
If you use:
•
•
•
•
3 bases = 1AA
Ex – CAT, AGC, TTT
43 = 64 combinations
More than enough for 20 amino acids.
Genetic Code
• Is based on triplets of bases.
• Has redundancy; some AA's have more than 1
code (codon).
• Proof - make artificial RNA and see what AAs
are used in protein synthesis (early 1960’s).
Codon
• A 3-nucleotide “word” in the Genetic Code.
• 64 possible codons known.
DNA vs RNA
Sugar –
Bases –
Backbones –
Size –
Use –
DNA
deoxyribose
ATGC
2
very large
genetic code
RNA
ribose
AUGC
1
small
varied
Codon Dictionary
• Start- AUG (Met)
• Stop- UAA
UAG
UGA
• 60 codons for the other 19 AAs.
For Testing:
• Be able to “read” a DNA or RNA message and
give the AA sequence.
• RNA Genetic Code Table will be provided.
Code Redundancy
• Third base in a codon shows "wobble”.
• First two bases are the most important in
reading the code and giving the correct AA.
The third base often doesn’t matter.
Code Evolution
• The genetic code is nearly universal.
• Ex: CCG = proline (all life)
• Reason - The code must have evolved very
early. Life on earth must share a common
ancestor.
Reading Frame and Frame Shift
• The “reading” of the code is every three
bases (Reading Frame)
– Ex: the red cat ate the rat
• Frame shift – improper groupings of the
bases
– Ex: thr edc ata tet her at
• The “words” only make sense if “read”
in this grouping of three.
Transcription
• Process of making RNA from a DNA template.
Transcription Steps
1.
2.
3.
4.
RNA Polymerase Binding
Initiation
Elongation
Termination
RNA Polymerase
• Enzyme for building RNA from RNA
nucleotides.
Binding
• Requires that the enzyme find the “proper”
place on the DNA to attach and start
transcription.
Binding
• Is a complicated process
• Uses Promoter Regions on the DNA (upstream
from the information for the protein)
• Requires proteins called Transcription Factors.
TATA Box
• Short segment of T,A,T,A
• Located 25 nucleotides upstream for the
initiation site.
• Recognition site for transcription factors to
bind to the DNA.
Transcription Factors
• Proteins that bind to DNA before RNA
Polymerase.
• Recognizes TATA box, attaches, and “flags” the
spot for RNA Polymerase.
Transcription Initiation Complex
• The complete assembly of transcription
factors and RNA Polymerase bound to the
promoter area of the DNA to be transcribed.
Initiation
• Actual unwinding of DNA to start RNA synthesis.
• Requires Initiation Factors.
Elongation
• RNA Polymerase untwists DNA 1 turn at a
time.
• Exposes 10 DNA bases for pairing with RNA
nucleotides.
Elongation
• Enzyme moves 5’
3’.
• Rate is about 60 nucleotides per second.
Comment
• Each gene can be read by sequential RNA
Polymerases giving several copies of RNA.
• Result - several copies of the protein can be
made.
Termination
• DNA sequence that tells RNA Polymerase to
stop.
• Ex: AATAAA
• RNA Polymerase detaches from DNA after
closing the helix.
Final Product
• Pre-mRNA
• This is a “raw” RNA that will need processing.
Modifications of RNA
1. 5’ Cap
2. Poly-A Tail
3. Splicing
5' Cap
• Modified Guanine nucleotide added to the 5'
end.
• Protects mRNA from digestive enzymes.
• Recognition sign for ribosome attachment.
Poly-A Tail
• 150-200 Adenine nucleotides added to the 3'
tail
• Protects mRNA from digestive enzymes.
• Aids in mRNA transport from nucleus.
Comment
• The head and tail areas often contain
“leaders” and “trailers”, areas of RNA that are
not read.
RNA Splicing
• Removal of non-protein coding regions of
RNA.
• Coding regions are then spliced back together.
Introns
• Intervening sequences (noncoding).
• Removed from RNA.
Exons
• Expressed sequences of RNA (coding).
• Translated into AAs.
Spliceosome
• Cuts out Introns and join Exons together.
• Made of snRNA and snRNPs.
snRNA
• Small Nuclear RNA.
• 150 nucleotides long.
• Structural part of spliceosomes.
snRNPs
•
•
•
•
("snurps")
Small Nuclear Ribonucleoprotiens
Made of snRNA and proteins.
Join with other proteins to form a
spliceosome.
Ribozymes
• RNA molecules that act as enzymes.
• Are sometimes Intron RNA and cause splicing
without a spliceosome.
Introns - Function
•
•
•
•
Left-over DNA (?)
Way to lengthen genetic message.
Old virus inserts (?)
Way to create new proteins.
Final RNA Transcript
Translation
• Process by which a cell interprets a genetic
message and builds a polypeptide.
Materials Required
• tRNA
• Ribosomes
• mRNA
Transfer RNA = tRNA
• Made by transcription.
• About 80 nucleotides long.
• Carries AA for polypeptide synthesis.
Structure of tRNA
• Has double stranded regions and 3 loops.
• AA attachment site at the 3' end.
• 1 loop serves as the Anticodon.
Anticodon
• Region of tRNA that base pairs to mRNA
codon.
• Usually is a compliment to the mRNA bases,
so reads the same as the DNA codon.
Example
• DNA - GAC
• mRNA - CUG
• tRNA anticodon - GAC
Comment
• "Wobble" effect allows for 45 types of tRNA
instead of 61.
• Reason - in the third position, U can pair with
A or G.
• Inosine (I), a modified base in the third
position can pair with U, C, or A.
Importance
• Allows for fewer types of tRNA.
• Allows some mistakes to code for the same AA
which gives exactly the same polypeptide.
Aminoacyl-tRNA Synthetases
•
•
•
•
Family of Enzymes.
Add AAs to tRNAs.
Active site fits 1AA and 1 type of tRNA.
Uses a “secondary genetic” code to load the
correct AA to each tRNA.
Ribosomes
• Two subunits made in the nucleolus.
• Made of rRNA (60%)and protein (40%).
• rRNA is the most abundant type of RNA in a
cell.
Large subunit
Proteins
rRNA
Both sununits
Large Subunit
• Has 3 sites for tRNA.
• P site: Peptidyl-tRNA site - carries the
growing polypeptide chain.
• A site: Aminoacyl-tRNA site -holds the
tRNA carrying the next AA to be
added.
• E site: Exit site
Translation Steps
1. Initiation
2. Elongation
3. Termination
Initiation
•
•
•
•
Brings together:
mRNA
A tRNA carrying the 1st AA
2 subunits of the ribosome
Initiation Steps:
1. Small subunit binds to the mRNA.
2. Initiator tRNA (Met, AUG) binds to mRNA.
3. Large subunit binds to mRNA. Initiator tRNA is
in the P-site.
Initiation
• Requires other proteins called "Initiation
Factors”.
• GTP used as energy source.
Elongation Steps:
1. Codon Recognition
2. Peptide Bond Formation
3. Translocation
Codon Recognition
• tRNA anticodon matched to mRNA codon in
the A site.
Peptide Bond Formation
• A peptide bond is formed between the new AA
and the polypeptide chain in the P-site.
• Bond formation is by rRNA acting as a ribozyme.
After bond formation
• The polypeptide is now transferred from the
tRNA in the P-site to the tRNA in the A-site.
Translocation
•
•
•
•
tRNA in P-site is released.
Ribosome advances 1 codon, 5’
3’.
tRNA in A-site is now in the P-site.
Process repeats with the next codon.
Comment
• Elongation takes 60 milliseconds for each AA
added.
Termination
• Triggered by stop codons.
• Release factor binds in the A-site instead of a
tRNA.
• H2O is added instead of AA, freeing the
polypeptide.
• Ribosome separates.
Polyribosomes
• Cluster of ribosomes all reading the same
mRNA.
• Another way to make multiple copies of a
protein.
Prokaryotes – How is this different?
Comment
• Polypeptide usually needs to be modified
before it becomes functional.
Examples
•
•
•
•
Sugars, lipids, phosphate groups added.
Some AAs removed.
Protein may be cleaved.
Join polypeptides together (Quaternary Structure).
Signal Hypothesis
• “Clue” on the growing polypeptide that causes
ribosome to attach to ER.
• All ribosomes are “free” ribosomes unless
clued by the polypeptide to attach to the ER.
Result
• Protein is made directly into the ER.
• Protein targeted to desired location (e.g.
secreted protein).
• “Clue” (the first 20 AAs are removed by
processing).
Mutations
• Changes in the genetic makeup of a cell.
• May be at chromosome (review chapter 15) or
DNA level
DNA or Point Mutations
• Changes in one or a few nucleotides in the
genetic code.
• Effects - none to fatal.
Types of Point Mutations
1. Base-Pair Substitutions
2. Insertions
3. Deletions
Base-Pair Substitution
• The replacement of 1 pair of nucleotides by
another pair.
Sickle Cell Anemia
Types of Substitutions
1. Missense - altered codons, still code for AAs
but not the right ones
2. Nonsense - changed codon becomes a stop
codon.
Question?
• What will the "Wobble" Effect have on
Missense?
• If the 3rd base is changed, the AA may still be
the same and the mutation is “silent”.
Comment
• Silent mutations may still have an effect by
slowing down the “speed” of making the
protein.
• Reason – harder to find some tRNAs than
others.
Missense Effect
• Can be none to fatal depending on where the
AA was in the protein.
• Ex: if in an enzyme active site = major effect.
If in another part of the enzyme = no effect.
Nonsense Effect
• Stops protein synthesis.
• Leads to nonfunctional proteins unless the
mutation was near the very end of the
polypeptide.
Sense Mutations
• The changing of a stop codon to a reading
codon.
• Result - longer polypeptides which may not be
functional.
• Ex. “heavy” hemoglobin
Insertions & Deletions
• The addition or loss of a base in the DNA.
• Cause frame shifts and extensive missense,
nonsense or sense mutations.
Question?
• Loss of 3 nucleotides is often not a problem.
• Why?
• Because the loss of a 3 bases or one codon
restores the reading frame and the protein
may still be able to function.
Mutagenesis
• Process of causing mutations or changes in
the DNA.
Mutagens
• Materials that cause DNA changes.
1. Radiation
ex: UV light, X-rays
2. Chemicals
ex: 5-bromouracil
Spontaneous Mutations
• Random errors during DNA replication.
Comment
• Any material that can chemically bond to DNA,
or is chemically similar to the nitrogen bases,
will often be a very strong mutagen.
What is a gene?
• A gene is a region of DNA that can be expressed
to produce a final functional product.
• The product can be a protein or a RNA molecule.
Protein vs RNA
• Protein – usually structure or enzyme for
phenotype
• RNA – often a regulatory molecule which will
be discussed in future chapters
Summary
•
•
•
•
Know Beadle and Tatum.
Know the central dogma.
Be able to “read” the genetic code.
Be able to describe the events of transcription
and translation.
• Be able to discuss RNA and protein processing.
• Be able to describe and discuss mutations.
• Be able to discuss “what is a gene?”.