Chapter 17From 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 DogmaDNA

RNA

Polypeptide

Transcription

Translation

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

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- UAAUAGUGA

• 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

• 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?”.