Proteins are polypeptide chains (may be one or more polypeptides, identical or different) composed of the 20 different amino acids.
Function of proteins
a) Structural (e.g.: microtubule protein, tubulin)
b) Enzymes (e.g.: luciferase)
c) Regulatory (e.g.: MalT, LacI)
d) Transport - Need energy for moving nutrients across the cellular membrane. (e.g.: Hemoglobin: a polypeptide with 4 subunits, 2 a and 2 b. It contains an iron cofactor heme which binds O2.)
Polypeptide chains fold to form an active site where the substrate is bound.
beta-Galactosidase:
- encoded by lacZ gene of lactose operon
- splits lactose into glucose and galactose
- easy to assay
- widely used in genetic analysis
Assaying for b-Galactosidase
b-Galactosidase cleaves:
- lactose into glucose (colorless) and galactose (colorless)
- ONPG into galactose (colorless) and ONP (ortho-nitro-phenol; yellow)
- X-gal into galactose (colorless) and indigo derivative (blue)
Analogs: molecules which mimic the true substrate
Competitive inhibitor: analog which blocks the active site so stops real substrate from getting in
Common features of the amino acid residue
Formation of a peptide bond. The peptide bond has a shared double bond making it flat and rigid. This does not allow rotation to occur.
An amino acid chain with:
- 2 amino acid residues is a dipeptide
- 3 amino acid residues is a tripeptide
- a few amino acid residues is an oligo-peptide
- many amino acid residues is a polypeptide
3D folding
Primary structure: linear order of subunits in a polymer (due to covalent bonding)
Secondary structure: initial folding of a polymer by hydrogen bonds
Hydrogen bonds are made between the C=O and N-H groups of different peptide bonds
Two structures allow maximum H-bonding:
- alpha-helix
- beta-sheet
The a-helix
a) 3.6 amino acids per turn
b) repeat every 5.4Å
c) diameter 2.3Å
d) H-bond to three ahead and three behind
The b-sheet
Hydrogen bonds go in two directions from each peptide bond. All possible H-bonds are made
Secondary structure: hydrogen bonding between peptide groups forming the a-helix and b-sheet
Tertiary structure: R-group interactions especially hydrophobic
Quaternary structure: assembly of the subunits
hydrophilic R-groups contact the water
hydrophobic R-groups cluster in the middle
Amino acid R-groups:
hydrophilic are acidic, basic and neutral
hydrophobic are aromatic and aliphatic
unique are glycine, cysteine, and proline
Greasy patches stick polypeptide subunits together.
Translation: the nucleic acid code (4 bases) read as codons and translated into a polypeptide (20 amino acids). Codon: group of 3 bases which code for a single amino acid
Principle of adaptor molecules
The cloverleaf structure of transfer RNA (tRNA)
Charging a tRNA molecule
Wobble:
64 codons
20 amino acids
How many different tRNA's?
tRNA anti-codon 1st base mRNA codon 3rd base
G C or U
U A or G
I C or U or A
C G only
A U only
Only 2 tRNA's at a time fit in the ribosome.
Start codon
AUG = methionine
- The first AUG is the start codon. (internal AUG codons also code for Met)
- Initiator tRNA carries formyl-methionine or fMet.
Shine Dalgarno sequence or ribosomal binding site (rbs) consensus sequence is AGGAGGGU. The ribosome will bind here in the start of translation. Translation begins at the first AUG following this. rbs binding to mRNA
Formation of the initiation complex
Initiation factors: oversee assembly of initiation complex
Elongation of the polypeptide chain
As move mRNA sideways:
a) tRNA #1 falls off
b) tRNA #2 moves into P-site
c) tRNA #3 enters A-site
P = peptide site
-holds tRNA with growing peptide chain
A = acceptor site
-accepts incoming tRNA
Elongation factors:
EF-T - helps install incoming tRNA in A-site
EF-G - moves things sideways ("translocation") uses GTP as energy
Stop codons:
UGA, UAG, UAA
no tRNA exists to read these stop codons
Proteins known as release factors recognize stop codons and chop the polypeptide chain off the final tRNA.
Eukaryotes have only one gene per mRNA.
Prokaryotes can have several genes per mRNA. This is polycistronic mRNA.
cistron = segment of DNA or RNA which encodes a single polypeptide chain
Open reading frame (ORF) = segment of DNA or RNA which has a start codon and goes on to the next stop codon
Polysome = several ribosomes read the same mRNA at once. Each ribosome has about 100-200 bases in between.
Coupled transcription-translation happens only in bacteria. The two events happen simultaneously.
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SIUC / College of Science / Microbiology / courses / MICR302
URL: http://www.micro.siu.edu/micro302/translation.html
Last updated: 09-Mar-99 / laa
