BIS101 Midterm 2: Protein, translation, and the genetic code

What do proteins do?

• biochemical catalysis

• provide structure for the cell

• control movement

• Transport of materials around the body

• Signaling

• Protection of the body and of individual cells

• Storage functions

Proteome

The entire complement of proteins present in a cell

Protein Structure

• Proteins are made of polypeptides

  • A polypeptide is a long chain of amino acids

• Amino acids have a free amino group, a free carboxyl group, and a side group (R)

• Amino acids are then joined by peptide bonds

  • -COOH of one AA is attached to the -NH2 of another

Levels of protein structure

• Primary structure

• Secondary structure

• Tertiary Structure

• quaternary structure

Primary Structure

amino acid sequence

Secondary structure

local structures involving H bonds of amino acids near one another, including alpha helices and beta sheets

Tertiary Structure

Overall 3D shape of polypeptide

Quaternary Structure

Shape of a protein containing more than one polypeptide chain

What do we need for translation?

• mRNA template

• Ribosomes, composed of polypeptide and RNA molecules

• tRNAs

• Amino acids, and Amino-acid activating enzymes

• Soluble proteins involved in polypeptide chain initiation, elongation, and termination

Ribosomes

• “machines” that contain multiple ribosomal RNAs and proteins

• translate mRNA in the 5’ to 3’ direction

• Read each triplet (3 bps) codon and assemble the amino acids in the order specified by the codons

tRNA

• adapters between amino acids and the codons in mRNA molecule

Aminoacyl-tRNA synthetase

• Add amino acids to the tRNAs

• Add the correct amino acid based on the anticodon the tRNA carries

• Highly accurate with an error rate of ~1:100,000

Specificity of tRNAs

• tRNA molecules must have the correct anticodon sequence

• tRNA molecules must be recognized by the correct aminoacyl-tRNA synthetase carrying the correct amino acid

• tRNA molecules must bind to the appropriate sites on the ribosome

A site

binds incoming tRNA carrying next amino acid to be added to the chain

P Site

binds tRNA to which growing peptide is attached

E site

binds departing uncharged tRNA

Prokaryotic Translation Initiation

• Have a small subunit

• Once small subunit binds, can build initiation complex

• Find start coden then find initiator tRNA (fMet) and bring it into P site

  • only time that you start off in the P site

• Then large subunit can assemble

• Then translation can begin

How does prokaryotic ribosome know where to start?

Shine-Dalgarno Sequence

• neaer 5’ end of mRNA, upstream of start codon

• This sequence is complementary to the 16s rRNA component of the small ribosomal subunit

• The ribosome P-site is positioned opposite the AUG start codon via this RNA: RNA pairing

Eukaryotic Translation Initiation

• Very similar but now have the 5’ cap so we know where to start

• A complex of the small subunit plus IF bind to the 5’ cap of mRNAs

• Scans for the first start odon from 5’ end

• initiation tRNA interacts with in IF and binds start codon at ribosome P site

• Large subunit binds, initiation is complete

How does eukaryotic ribosome know where to start?

• find 5’ cap

• scan along transcript

• most of the time use first AUG from 5’ end

• but higher efficiency of using AUG is when it’s located in Kozak sequence

  • even if there’s another AUG closer to 5’ cap

Elongation (for both prokaryotes and eukaryotes)

• Aminoacyl-tRNA enters A site of ribosome, anticodon of tRNA must pair with codon in A site

• Transfer of growing polypeptide from tRNA in P site to tRNA in A site

• Translocate ribosome along 3 nucleotides toward 3’ end of transcript

• Growing chain is in P site, uncharged tRNA in E site can then depart

Polypeptide Chain Termination

• Polypeptide chain termination occurs when stop codon enters A site of ribosome

  • UAA, UAG, UGA

• when stop codon is encountered, a release factors binds to the A site, and the discharged tRNA in the E site departs

• ribosome-mRNA-tRNA complex disassembles

Reversion Mutation

a genetic change that restores the original wild-type phenotype (appearance or function) in a mutant organism that previously acquired a forward mutation

Properties of the Genetic Code

• It is composed of nucleotides triplets (codons)

• It is nonoverlapping

• It is comma-free

  • there are no pauses, punctuation, or gaps between codons

• It is degenerate (more than one codon codes for the same amino acid)

• It is ordered

• It contains start and stop codons

• It is nearly universal

Third Base Wobble

• There are 61 codons that specify amino acids, but only 30-50 tRNAs in most genomes

• This means there aren’t enough tRNAs for each to have a unique codon

• Relaxation of the strict complementary base-pairing rules at the third base of the codon is called third base wobble

• this permits a single tRNA to bind more than one codon in the mRNA

Post-Translational Processes

• Protein Folding

• Chemical modification of amino acids

• Cleavage of polypeptides

Protein Folding

• Primary, Secondary, etc structures

• Proteins called chaperones help other proteins fold correctly

  • can help refold if they unfold

  • can sequester misfolded proteins

• if a protein cant fold, its a loss of function

• if misfolded proteins build up, they cause problems

Chemical modification of amino acids

Adding a new chemical group to a particular AA

Cleavage of Polypeptides

• removal of fMet from N-terminus in bacteria

• Polypeptides may be cleaved into multiple segments that have separate functions or that aggregate to form a functional protein