From Gene to Protein

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

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.

Genetic Code u Is based on triplets of bases. u Has redundancy; some AA's have more than 1 code. u 20 amino acids: 64 codons

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. u Only one strand is used as a template.

Where in the cell does transcription take place? u Eukaryotes: nucleus u Prokaryotes: cytoplasm

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 (start region) on the DNA (upstream from the information for the protein)

Initiation u Actual unwinding of DNA to start RNA synthesis.

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.

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.

Result

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

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

Where in the cell does translation take place? u Eukaryotes and prokaryotes: cytoplasm because that’s where the ribosomes are located.

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

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

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.

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 u tRNA in A-site is now in the P-site. u Process repeats with the next codon.

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.

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).