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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:
DNA RNA
Sugar – deoxyribose ribose
Bases – ATGC AUGC
Backbones – 2 1
Size – very large small
Use – genetic code 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. RNA Polymerase Binding
2. Initiation
3. Elongation
4. 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