Chapter 15 From Genes to Proteins

Question? u How does DNA control a cell? u By controlling Protein Synthesis. u Proteins are the link between genotype and phenotype.

For tests: u Name(s) of experimenters u Outline of the experiment u Result of the experiment and its importance

Archibald Garrod u Suggested genes control enzymes that catalyze chemical processes in cells. u Inherited Diseases - “inborn errors of metabolism” where a person can’t make an enzyme.

Example u Alkaptonuria - where urine turns black after exposure to air. u Lacks - an enzyme to metabolize alkapton.

George Beadle and Edward Tatum u Worked with Neurospora and proved the link between genes and enzymes. Neurospora Pink bread mold

Experiment u Grew Neurospora on agar. u Varied the nutrients. u Looked for mutants that failed to grow on minimum agar.

Results u Three classes of mutants for Arginine Synthesis. u Each mutant had a different block in the Arginine Synthesis pathway.

Conclusion u Mutations were abnormal genes. u Each gene dictated the synthesis of one enzyme. u One Gene - One Enzyme Hypothesis.

Current Hypothesis u One Gene - One Polypeptide Hypothesis (because of 4 th degree structure).

Central Dogma DNA Transcription RNA Translation Polypeptide

Explanation u DNA - the Genetic code or genotype. u RNA - the message or instructions. u Polypeptide - the product for the phenotype.

Genetic Code u Sequence of DNA bases that describe which Amino Acid to place in what order in a polypeptide. u The genetic code gives the primary protein structure.

Code Basis If you use: u 1 base = 1 amino acid u 4 bases = 4 amino acids u 4 1 = 4 combinations, which are not enough for 20 AAs.

If you use: u 2 bases = 1 amino acid u 4 2 = 16 amino acids u Still not enough combinations.

If you use: u 3 bases = 1AA u 4 3 = 64 combinations u More than enough for 20 amino acids.

Genetic Code u Is based on triplets of bases. u Has redundancy; some AA's have more than 1 code. u Proof - make artificial RNA and see what AAs are used in protein synthesis (early 1960’s).

Codon u A 3-nucleotide “word” in the Genetic Code. u 64 possible codons known.

Codon Dictionary u Start- AUG (Met) u Stop- UAA UAG UGA u 60 codons for the other 19 AAs.

For Testing: u Be able to “read” a DNA or RNA message and give the AA sequence. u RNA Genetic Code Table will be provided.

Code Redundancy u Third base in a codon shows "wobble”. u 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 u The genetic code is nearly universal. u Ex: CCG = proline (all life) u Reason - The code must have evolved very early. Life on earth must share a common ancestor.

Reading Frame and Frame Shift u The “reading” of the code is every three bases (Reading Frame) u Ex: the red cat ate the rat u Frame shift – improper groupings of the bases u Ex: thr edc ata tat her at u The “words” only make sense if “read” in this grouping of three.

Transcription u Process of making RNA from a DNA template.

Transcription Steps 1. RNA Polymerase Binding 2. Initiation 3. Elongation 4. Termination

RNA Polymerase u Enzyme for building RNA from RNA nucleotides.

Binding u Requires that the enzyme find the “proper” place on the DNA to attach and start transcription.

Binding u Is a complicated process u Uses Promoter Regions on the DNA (upstream from the information for the protein) u Requires proteins called Transcription Factors.

TATA Box u Short segment of T,A,T,A u Located 25 nucleotides upstream for the initiation site. u Recognition site for transcription factors to bind to the DNA.

Transcription Factors u Proteins that bind to DNA before RNA Polymerase. u Recognizes TATA box, attaches, and “flags” the spot for RNA Polymerase.

Transcription Initiation Complex u The complete assembly of transcription factors and RNA Polymerase bound to the promoter area of the DNA to be transcribed.

Initiation u Actual unwinding of DNA to start RNA synthesis. u Requires Initiation Factors.

Elongation u RNA Polymerase untwists DNA 1 turn at a time. u Exposes 10 DNA bases for pairing with RNA nucleotides.

Elongation u Enzyme moves 5’ 3’. u Rate is about 60 nucleotides per second.

Comment u Each gene can be read by sequential RNA Polymerases giving several copies of RNA. u Result - several copies of the protein can be made.

Termination u DNA sequence that tells RNA Polymerase to stop. u Ex: AATAAA u RNA Polymerase detaches from DNA after closing the helix.

Final Product u Pre-mRNA u This is a “raw” RNA that will need processing.

Modifications of RNA 1. 5’ Cap 2. Poly-A Tail 3. Splicing

5' Cap u Modified Guanine nucleotide added to the 5' end. u Protects mRNA from digestive enzymes. u Recognition sign for ribosome attachment.

Poly-A Tail u Adenine nucleotides added to the 3' tail u Protects mRNA from digestive enzymes. u Aids in mRNA transport from nucleus.

Comment u The head and tail areas often contain “leaders” and “trailers”, areas of RNA that are not read. u Similar to leaders or trailers on cassette tapes.

u Let’s see Transcription in motion… u ctive/media/DNAi_transcriptio n_vo2-lg.mov ctive/media/DNAi_transcriptio n_vo2-lg.mov

RNA Splicing u Removal of non-protein coding regions of RNA. u Coding regions are then spliced back together.

Introns u Intervening sequences. u Removed from RNA.

Exons u Expressed sequences of RNA. u Translated into AAs.

Spliceosome u Cut out Introns and join Exons together. u Made of snRNA and snRNP.

snRNA u Small Nuclear RNA. u 150 nucleotides long. u Structural part of spliceosomes.

snRNPs u ("snurps") u Small Nuclear Ribonucleoprotiens u Made of snRNA and proteins. u Join with other proteins to form a spliceosome.

Result

Ribozymes u RNA molecules that act as enzymes. u Are sometimes Intron RNA and cause splicing without a spliceosome.

Introns - Function u Left-over DNA (?) u Way to lengthen genetic message. u Old virus inserts (?) u Way to create new proteins.

Final RNA Transcript

Alternative Splicing u The RNA can be spliced into different mRNA’s. u Each different mRNA produces a different polypeptide. u Ex. – variable regions of antibodies.

Another Example

u Bcl-X L – inhibits apoptosis u Bcl-X S – induces apoptosis u Two different and opposite effects!!

DSCAM Gene u Found in fruit flies u Has 100 potential splicing sites. u Could produce 38,000 different polypeptides u Many of these polypeptides have been found

Commentary u Alternative Splicing is going to be a BIG topic in Biology. u About 60% of genes are estimated to have alternative splicing sites. u One gene does not equal one polypeptide.

Translation u Process by which a cell interprets a genetic message and builds a polypeptide.

Materials Required u tRNA u Ribosomes u mRNA

Transfer RNA = tRNA u Made by transcription. u About 80 nucleotides long. u Carries AA for polypeptide synthesis.

Structure of tRNA u Has double stranded regions and 3 loops. u AA attachment site at the 3' end. u 1 loop serves as the Anticodon.

Anticodon u Region of tRNA that base pairs to mRNA codon. u Usually is a compliment to the mRNA bases, so reads the same as the DNA codon.

Example u DNA - GAC u mRNA - CUG u tRNA anticodon - GAC

Comment u "Wobble" effect allows for 45 types of tRNA instead of 61. u Reason - in the third position, U can pair with A or G. u Inosine (I), a modified base in the third position can pair with U, C, or A.

Importance u Allows for fewer types of tRNA. u Allows some mistakes to code for the same AA which gives exactly the same polypeptide.

Aminoacyl-tRNA Synthetases u Family of Enzymes. u Add AAs to tRNAs. u Active site fits 1AA and 1 type of tRNA. u Uses a “secondary genetic” code to load the correct AA to each tRNA.

Ribosomes u Two subunits made in the nucleolus. u Made of rRNA (60%)and protein (40%). u rRNA is the most abundant type of RNA in a cell.

Large subunit Proteins rRNA

Both sununits

Large Subunit u Has 3 sites for tRNA. u P site: Peptidyl-tRNA site - carries the growing polypeptide chain. u A site: Aminoacyl-tRNA site - holds the tRNA carrying the next AA to be added. u E site: Exit site

Translation Steps 1. Initiation 2. Elongation 3. Termination

Initiation u Brings together: u mRNA u A tRNA carrying the 1st AA u 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 u Requires other proteins called "Initiation Factors”. u GTP used as energy source.

Elongation Steps: 1. Codon Recognition 2. Peptide Bond Formation 3. Translocation

Codon Recognition u tRNA anticodon matched to mRNA codon in the A site.

Peptide Bond Formation u A peptide bond is formed between the new AA and the polypeptide chain in the P-site. u Bond formation is by rRNA acting as a ribozyme

After bond formation u The polypeptide is now transferred from the tRNA in the P-site to the tRNA in the A-site.

Translocation u tRNA in P-site is released. u Ribosome advances 1 codon, 5’ 3’. u tRNA in A-site is now in the P-site. u Process repeats with the next codon.

Comment u Elongation takes 60 milliseconds for each AA added.

Termination u Triggered by stop codons. u Release factor binds in the A-site instead of a tRNA. u H 2 O is added instead of AA, freeing the polypeptide. u Ribosome separates.

u Let’s see Translation in motion… u ctive/media/DNAi_translation _vo2-lg.mov ctive/media/DNAi_translation _vo2-lg.mov

Polyribosomes u Cluster of ribosomes all reading the same mRNA. u Another way to make multiple copies of a protein.

Prokaryotes

Comment u Polypeptide usually needs to be modified before it becomes functional.

Examples u Sugars, lipids, phosphate groups added. u Some AAs removed. u Protein may be cleaved. u Join polypeptides together (Quaternary Structure).

Signal Hypothesis u “Clue” on the growing polypeptide that causes ribosome to attach to ER. u All ribosomes are “free” ribosomes unless clued by the polypeptide to attach to the ER.

Result u Protein is made directly into the ER. u Protein targeted to desired location (e.g. secreted protein). u “Clue” (the first 20 AAs are removed by processing).

Mutations u Changes in the genetic makeup of a cell. u May be at chromosome or DNA level

Chromosome Alterations u Deletions u Duplications u Inversions u Translocations

General Result u Loss of genetic information. u Position effects: a gene's expression is influenced by its location to other genes.

Evidence of Translocation Translocations

Cri Du Chat Syndrome u Part of p arm of #5 has been deleted. u Good survival. u Severe mental retardation. u Small sized heads common.

Philadelphia Chromosome u An abnormal chromosome produced by a translocation of portions of chromosomes 9 and 22. u Causes chronic myeloid leukemia.

Mutation types - Cells u Somatic cells or body cells – not inherited u Germ Cells or gametes - inherited

DNA or Point Mutations u Changes in one or a few nucleotides in the genetic code. u Effects - none to fatal.

Types of Point Mutations 1. Base-Pair Substitutions 2. Insertions 3. Deletions

Base-Pair Substitution u The replacement of 1 pair of nucleotides by another pair.

Sickle Cell Anemia

u Lets see how this mutation will affect the cell… u ctive/media/DNAi_sicklecell- lg.mov ctive/media/DNAi_sicklecell- lg.mov

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? u What will the "Wobble" Effect have on Missense? u If the 3rd base is changed, the AA may still be the same and the mutation is “silent”.

Missense Effect u Can be none to fatal depending on where the AA was in the protein. u Ex: if in an active site - major effect. If in another part of the enzyme - no effect.

Nonsense Effect u Stops protein synthesis. u Leads to nonfunctional proteins unless the mutation was near the very end of the polypeptide.

Sense Mutations u The changing of a stop codon to a reading codon. u Result - longer polypeptides which may not be functional. u Ex. “heavy” hemoglobin

Insertions & Deletions u The addition or loss of a base in the DNA. u Cause frame shifts and extensive missense, nonsense or sense mutations.

Question? u Loss of 3 nucleotides is often not a problem. u Why? u Because the loss of a 3 bases or one codon restores the reading frame and the protein may still be able to function.

Mutagenesis u Process of causing mutations or changes in the DNA.

Mutagens u Materials that cause DNA changes. 1. Radiation ex: UV light, X-rays 2. Chemicals ex: 5-bromouracil

Spontaneous Mutations u Random errors during DNA replication.

Comment u Any material that can chemically bond to DNA, or is chemically similar to the nitrogen bases, will often be a very strong mutagen.

Summary u Know Beadle and Tatum. u Know the central dogma. u Be able to “read” the genetic code. u Be able to describe the events of transcription and translation.

Summary u Be able to discuss RNA and protein processing. u Be able to describe and discuss mutations.