Protein Synthesis Notes

Protein Synthesis: Overview Transcription: synthesis of mRNA under the direction of DNA. Translation: actual synthesis of a polypeptide under the direction of mRNA.

Transcription Process of making RNA from a DNA template.

Transcription Steps RNA Polymerase Binding Initiation Elongation Termination

RNA Polymerase: Enzyme for building RNA from RNA nucleotides. Prokaryotes - 1 type Eukaroyotes- 3 types

RNA Polymerase Binding: Requires that the enzyme find the “proper” place on the DNA to attach and start transcription – the Promoter Region.

RNA Polymerase Binding Needs: Promoter Regions on the DNA. Transcription Factors.

Promoters Regions of DNA where RNA Polymerases can bind. About 100 nucleotides long. Include initiation site and recognition areas for RNA Polymerase.

Promoter region at the front of the gene to be transcribed.

TATA Box Short segment of T,A,T,A repeated. 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. Each factor recognizes a different area, such as the TATA box. They each bind to area to “flag” 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.

Transcription Complex Only when all transcription factors have been picked up by and bonded to the RNA Polymerase, can transcription begin.

Initiation Actual unwinding of DNA to start RNA transcription. Requires Initiation Factors.

Getting Transcription started is complicated. Gives many ways to control which genes are decoded and which proteins are synthesized.

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

Elongation Enzyme builds 5’ 3’. That means it transcribes the 3 > 5’ strand – the is called the anti- sense strand. Rate is about 60 nucleotides per second.

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

At the End of Transcription: We have Pre-mRNA This is a “raw” RNA that will need processing (or Modification).

Modifications of RNA 5’ Cap Poly-A Tail Splicing

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

This mRNA will be threaded through a ribosome like film through a projector. The 5’ cap protects the leading edge of the mRNA from wear and tear.

Poly-A Tail 150-200 Adenine nucleotides added to the 3' tail Protects mRNA from digestive enzymes. Aids in mRNA transport from nucleus.

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

Introns Intervening sequences. Removed from RNA.

Exons Expressed sequences of RNA. • Translated into AAs.

Result

Introns - Function Left-over DNA (?) Way to lengthen genetic message. Old virus inserts (?) Way to create new proteins. Help reduce likelihood of accidental damaging mutation.

mRNA modification 1) 5’ cap: modified guanine; protection; recognition site for ribosomes 2) 3’ tail: poly(A) tail (adenine); protection; recognition; transport 3) RNA splicing: exons (expressed sequences) kept,introns (intervening sequences) spliced out; spliceosome

Transcription Movie:

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

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.

Both subunits

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 Initiation Elongation Termination

Initiation Brings together: mRNA A tRNA carrying the 1st AA 2 subunits of the ribosome

Initiation Steps: Small subunit binds to the mRNA. Initiator tRNA (Met, AUG) binds to mRNA. Large subunit binds to mRNA. Initiator tRNA is in site the P-

Initiation Requires other proteins called "Initiation Factors”. GTP used as energy source.

Elongation Steps: Codon Recognition Peptide Bond Formation 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. Elongation takes 60 milliseconds for each AA added.

Termination Triggered by stop codons. Release factor binds in the instead of a tRNA. A-site H2O is added instead of AA, freeing the polypeptide. Ribosome separates.

Translation

Protein Structure

Size and Shape Compariso n of Proteins

Levels of Protein Structure

Amino Acids

Peptide Bonds Proteins are formed by creating peptide bonds between individual amino acids. Remove water Called dehydration

Peptide Bonds

20 Amino Acids

Some Amino Acids have/are: A Negative Charge A Positive Charge Uncharged & Polar Nonpolar

Amino Acids Hydrophilic Hydrophobic

Secondary (2°) Structure Folding into α- helix or β- sheets

α-Helix

Myoglobin

β-sheet

β - Sheets 2 kinds: Parallel Antiparallel Parallel Antiparallel

β - Sheets COXSACKIE VIRUS AND ADENOVIRUS RECEPTOR Antiparallel

Often both structures are found in the same molecule:

Tertiary (3°) Structure

3° Structure 3-D Conformation of Protein contain “domains” (~50-350 aa) that fold and function independently may contain many domains

Domain 1

Domain 2

Domain 3

Domain 4

Quarternary (4°) Structure

4° Structure association of polypeptides into multi- subunit protein

Catalase Quaternary Structure

Protein Functions Structural Regulatory Enzyme Transport