Download By controlling Protein Synthesis

Chapter 17
From Gene to Protein
• Describe the structure of DNA. What
is its elemental makeup? Name the
subunit that makes up DNA. What
components make up the DNA
molecule? How are the two strands
related and connected? How are they
different?
Question?
• How does DNA control a cell?
• By controlling Protein Synthesis.
• Proteins are the link between genotype
and phenotype.
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.
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 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.
• 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
DNA
Sugar – deoxyribose
Bases – ATGC
Backbones – 2
Size – very large
Use – genetic code
RNA
ribose
AUGC
1
small
varied
Codon Dictionary
• Start- AUG (Met)
• Stop- UAA
UAG
UGA
• 60 codons for the other 19 AAs.
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 tat 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.
• Similar to leaders or trailers on
cassette tapes.
RNA Splicing
• Removal of non-protein coding regions
of RNA.
• Coding regions are then spliced back
together.
Introns
• Intervening sequences.
• Removed from RNA.
Exons
• Expressed sequences of RNA.
• Translated into AAs.
Spliceosome
• Cut out Introns and join Exons
together.
• Made of snRNA and snRNP.
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
• If a segment of DNA is 5’-TGA AGA
CCG-3’, the resulting RNA strand
from the transcription of this would
read:
– 5’-TGA AGA CCG-3’
– 5’-UGA AGA CCG-3’
– 3’- CGG UCU UCA- 5’
– 3’- ACU UCU GGC- 5’
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
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).
• The flow of genetic information from
DNA to protein in eukaryotic cells is
called the central dogma of biology.
– Write the central dogma of biology as a flow
chart
– What role does each of these structures play
in protein synthesis?
• DNA
• mRNA
• RNA polymerase
• Spliceosomes
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 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.
Summary
• Be able to discuss RNA and protein
processing.
• Be able to describe and discuss
mutations.
• Be able to discuss “what is a gene”.