1. Chapter 15
DNA to Protein

2. 15.1 The Nature of Genes
Archibald Garrod-investigated genetic disorders
Found that patients with alkaptonuria produced urine that contained homogentisic acid (when urine was exposed to the air, it turned black).
Garrod concluded that these patients lacked an enzyme to catalyze the breakdown of the acid.
Beadle & Tatum- set out to create Mendelian mutations in chromosomes and then study the effects.
Worked with bread mold (fungus); exposed mold to x-ray to damage the DNA.
Isolated growth deficient mutants
Identified the deficiencies (ex: added arginine to the medium and this allowed the mutants to grow)
Results: each mutant had a defect in a single enzyme, caused by a mutation at a single site on one chromosome.
“One gene/one polypeptide”- genes produce their effects by specifying the structure of enzymes and that each gene encodes the structure of one enzyme.

3. 15.1 The Nature of Genes The Central Dogma: DNA  RNA  Protein
Same basic mechanism used from bacteria to humans
Transcription ( DNA to mRNA)
Translation (RNA to protein)
Cells use RNA to make protein
The site of protein synthesis is the ribosome (three specific sites P, A and E)
Six types of RNA:
mRNA (messenger)- transcribes the DNA message in the nucleus and brings it to the ribosome.
tRNA (transfer)- transports amino acids to the ribosome and positions them in the correct place on the polypeptide chain.
rRNA (ribosomal)- provides the site where polypeptide are assembled.

4. 15.1 cont’d
Small nuclear RNA- part of the machinery involved in processing eukaryotic pre-mRNA.
SRP RNA- signal recognition particle- mediates process of proteins synthesized by ribosomes on the rough ER.
Small RNAs- micro-RNA and small interfering RNA are both involved in gene expression (Chapter 16)

5. 15.2 The Genetic Code
How does the order of nucleotides in a DNA molecule encode the information that specifies the order of amino acids in a protein?
1961 Francis Crick- hypothesized that blocks of information (codons) made up the genetic code and that each codon corresponds to an amino acid in a protein.
Crick’s hypothesis stated the 3 nucleotides made up a codon because 43=64 possible combinations of A, T, C and G which is more than enough to code for 20 amino acids.
The Experiment
Used chemicals to eliminate one, two or three nucleotides from viral DNA and then observed if gene down stream was transcribed correctly.
When one or two nucleotides were deleted, the “reading frame” was shifted and gene was not transcribed correctly.
When three were deleted, correct reading frame was restored.

6. 15.2 The Genetic Code
Between 1961 & 1966 the genetic code was broken (table 15.1 p. 283)
61 of the 64 possible codons are used to specify amino acids
Three codon, UAA, UGA and UAG are reserved for “stop” signals
AUG is reserved for the “start” signal
Code is universal (practically)
AGA codes for arginine in bacteria, humans and all other organisms studied.
Evidence of common evolutionary heritage.
Gene transfer is possible.
Allows for the mass production of commercial products. Ex: insulin
Mitochondrial genomes are not quite the same as the “universal code”

7. 15.3 Bacteria vs Eukaryotic Gene Expression
Eukaryotes possess introns, prokaryotes do not.
Bacterial transcripts possess information for several genes. Eukaryotic transcripts possess information for one gene.
Bacteria begin translation before transcription is even complete.
Eukaryotic transcripts are modified:
5 ̷̷ end cap
3 ̷ end poly-A tail
Introns cut out, exons spliced together
5. Ribosomes in eukaryotes are larger than bacterial ribosomes.

8. 15.4 Transcription
DNA  mRNA = Transcription RNA polymerases are the primary enzymes responsible for this process 1. Promoters- process starts at RNA polymerase binding sites (promoters) on the DNA template strand. Promoters are short sequences that are not transcribed by the polymerase that binds to them. Ex: TATA box- 25 nucleotides upstream from target gene. There are strong promoters (frequently initiated) and weak promoters (less frequently initiated). 2. Initiation- RNA polymerase binds to promoter. Other eukaryotic factors bind one after the another assembling a large transcription complex. RNA polymerase begins to unwind DNA and sets the stage for the assembly of the RNA chain.

9. 15.3 Transcription
3. Elongation- transcription usually starts with ATP or GTP. Growth will occur in the 5 ̷  3 ̷ direction. No primer is required; transcription bubble is created (RNA polymerase, DNA and growing RNA transcript). 12 bases of transcript form a helix with DNA template in order to stabilize it. Bubble moves down DNA at about 50 nucleotides/second.
Termination- at the end of genes are “stop” sequences causing transcription to stop, DNA/RNA hybrid to dissociate, RNA polymerase to release DNA and DNA to rewind. A GC hairpin is created to stop process.
Modifications- 5 ̷̷ cap (protects against nucleases and phosphatases in cytoplasm)
3 ̷ poly-A tails -250 nucleotides added to 3 ̷ end (protects against degradation by nucleases)
Tutorial 12.1 Transcription

10. 15.5 Eukaryotic pre-mRNA Splicing
A typical gene is not simply a straight sequence of DNA, it is fragmented.
The DNA units that code for a particular protein are called exons.
Pieces of DNA that do not code for a particular protein are called introns.
When DNA is transcribed, a primary transcript is produced.
Small nuclear ribonucleoprotein particles (snRNP’s) associate with other proteins to form spliceosomes. Within spliceosomes, introns, are folded and cut out; exons are then spliced together.
Animation - Exon Shuffling

11. 15.6 The Structure of tRNA and Ribosomes
Each amino acid must be attached to a tRNA with the correct anitcodon for protein synthesis to occur. This is accomplished by activating enzymes called aminoacyl-tRNA synthetases. There is one enzyme for each of the 20 amino acids.
tRNA molecules are bifunctional; they need to interact with amino acids and mRNA molecules.
There is an acceptor stem, where amino acid binds
There is the anticodon loop that base pairs with mRNA
There is a charging reaction that is catalyzed by the
enzymes specific to their amino acid

12. 15.6 cont’d
Ribosomes have multiple tRNA binding sites. In order for it to form peptide bonds, the ribosome must be able to bind two tRNA molecules at the same time.
The P site binds to the tRNA attached to the growing peptide chain
The A site binds to the tRNA carrying the next amino acid to be added
The E site binds to the tRNA that carried the previous amino acid added

13. 15.7 Translation
Translation- the assembly of proteins on the ribosomes using mRNA to specify the order of amino acids.
Begins when the initial portion of an mRNA molecule binds to a tRNA molecule on a ribosome (only one codon on mRNA is exposed at the polypeptide making site at a time) ex: CCU=codon
GGA = anticodon
There are about 45 kinds of tRNA molecules (not 64) because the third base pair allows some “wobble” (some tRNA molecules recognize more than one codon).

14. 15.7 Translation
1. Initiation- tRNA binds to small ribosomal subunit at the P site (proteins called initiation factors position tRNA). P stands for peptidyl (where peptide bonds will form). This complex binds the anticodon on tRNA to AUG on mRNA. 2. Elongation- large ribosomal subunit binds, exposing the mRNA codon adjacent to the AUG codon. When a tRNA molecule with the appropriate anticodon appears, elongation factors assist in binding, to the mRNA at the A site (aminoacyl).

15. 15.7 Translation
Translocation- ribosomes move three more nucleotides along the mRNA This relocates initial tRNA to the E site (exit) and ejects it from the ribosome, repositioning growing polypeptide chain to the P site and exposes the next codon at the A site.
Termination- nonsense codon is reached (UAA) and polypeptide is released.
Animation: How Translation Works

16. 15.7 cont’d
In eukaryotes, translation can occur in either the cytoplasm or on the rough ER.
Proteins that are translated on the rough ER are targeted there based on their own initial amino acid sequence.
The polypeptide that starts with a short series of amino acids called signal sequence is specifically recognized and bound by a cytoplasmic complex of proteins called the signal recognition particle (SRP).
The binding of the ER receptor to the signal/SRP complex holds the ribosome engaged in translation to the ER membrane.
The production of a protein in this manner is the beginning of the protein- trafficking pathway that can lead to other intracellular targets, incorporation into plasma membrane or release out of the cell.

17. 15.9 Mutation: Altered Genes
Types of Mutations in Genes
1. Point mutation- alteration in a single base pair in a coding sequence.
Base substitution- one base for another. Ex: C instead of T
Missense mutation- substitution that changes an amino acid in a protein
Nonsense mutation- base is changed such that the transcribed codon is converted to a stop codon.
2. Frameshift mutation- deletion or insertion-causes the wrong three base groupings to be read.
Ex: the fat cat ate the rat
the atc ata tet her at

18. 15.9 cont’d
Trinucleotide Repeats- nucleotide sequences repeated hundreds of times. Function is unknown even though they seem common in human genes.
Ex: Huntington disease- contains a triplet sequence of DNA that is repeated and this repeat unit has expanded in the disease allele relative to the normal allele.
Mutations in Chromosomes
1. Deletion- results in to few chromosomes
Ex: Turner Syndrome-missing the second sex chromosome-XO
2. Duplications- results in too many chromosomes
Ex: Down Syndrome = and extra 21st chromosome

19. 15.9 Inversions- orientation of part of a chromosome is reversed.
Translocation- segment of one chromosome becomes part of another.
Mutations are the starting point of evolution. Mutations give rise to variation within a species.