Genetic Code
- relationship between sequence of nucleotides in DNA / RNA and sequence of amino acids in a protein
- 64 (4^3) possible codons yet only 20 amino acids (AAs)
- all AAs except methionine (Met) and tryptophan (Trp) specified by more than one codon, but one codon never specifies more than one AA
Genetic code pt 2
- for AAs with multiple codons, third base is most variable
- genetic code is almost universal
- same codons assigned to the same amino acids and to the same START and STOP signals in the vast majority of genes in animals, plants, and microbes
- exceptions: some fungi and protozoa; mitochondria
Exceptions to Genetic Code
common exception is to assign some of the three STOP codons to an amino acid
exceptions in mitochondria:
- e.g. mitochondria from animal cells use UGA to encode tryptophan (rather than STOP)
has implications for transferring of mitochondrial genes to nuclear genome
- cytosolic protein- synthesizing machinery reading a mitochondrial gene will always STOP when it should be inserting a tryptophan!
Genetic Code and Reading Frame
- loss or gain of bases (deletions, insertions) that shift the reading frame (frame shift mutations) can lead to novel proteins that are beneficial, or they can be disastrous …
Redundancy in the Genetic Code and tRNAs
- several different codons can specify the same AA
what about the specificity of tRNAs? - some AAs have more than one tRNA
- some tRNAs need accurate base-pairing at only the first two bases of a codon
- can tolerate mismatch (‘wobble’) at third position
How does an mRNA codon specify an amino acid?
- Francis Crick proposed that an ‘adapter molecule’ held amino acids in place while interacting directly and specifically with a codon in mRNA.
- Transfer RNA is the Adapter
- each amino acid has its own aminoacyl tRNA synthetase
wobble hypothesis:
the anticodon of tRNAs can still bind successfully to a codon whose third position requires a nonstandard base pairing
Loading tRNA with amino acid: aminoacyl-tRNA synthetase
- total of 20 aminoacyl-tRNA synthetases
- each synthetase must recognize its amino acid plus all anticodons that recognize that amino acid
- hydrolysis of ATP will be coupled to attachment of amino acid to tRNA
- combined action of tRNA and synthetases ensures that each mRNA codon is matched to correct amino acid
‘charging’ of tRNA (e.g. tRNA specific to leucine)
- active site binds ATP & amino acid
- leucine bound to AMP now ‘activated
- activated amino acid
transferred to tRNA (tRNA specific to leucine) - finished aminoacyl tRNA
ready for translation
Components of Ribosomes (eukaryotic)
large subunit:
catalyzes formation of peptide bonds
small subunit:
matches tRNAs to codons
Overview of Mechanism of Translation
translation begins …
- when the anticodon of a ‘charged’ tRNA binds to a codon in mRNA
translation ends …
- when that amino acid forms a peptide bond with growing chain
Translation: Initiation
- The small ribosomal subunit binds to the mRNA molecule
- The initiation complex forms when the initiator tRNA carrying methionine binds to the start codon (AUG) on the mRNA
- Then, the large ribosomal subunit joins the complex, forming a functional ribosome.
Translation: Translocation
- After each peptide bond formation, the ribosome advances to the next codon along the mRNA, causing the ribosome to move relative to the mRNA
- This movement shifts the tRNAs from the A (aminoacyl) site to the P (peptidyl) site and then to the E (exit) site
- The tRNA is released from the E site, and the process repeats as the ribosome moves to the next codon.
Translation: Elongation
- the ribosome moves along the mRNA, reading the codons one by one.
- Aminoacyl-tRNA molecules carrying specific amino acids enter the ribosome, and their anticodons base-pair with complementary codons on the mRNA
- Peptide bonds form between adjacent amino acids, creating a growing polypeptide chain
Translation: termination
- When a stop codon (UAA, UAG, or UGA) is encountered on the mRNA, it signals the termination of protein synthesis
- Release factors bind to the stop codon, causing the ribosome to release the completed polypeptide chain
- The ribosome subunits dissociate from the mRNA, and the newly synthesized protein is released into the cytoplasm for further processing or targeting to its functional location.
what is a release factor in translation
alters catalytic activity, causing addition of a water rather then forming a peptide bond
Is the ribosome an enzyme or a ribozyme?
- A, P and E sites are primarily rRNA
- catalytic site where peptide bond is formed between P and A sites in large subunit is formed entirely by RNA, not protein
- ribosomal proteins are mostly on surface, helping to create and maintain shape of RNA core
ribozyme definition
RNA molecule with a well defined tertiary structure that enables it to catalyze a chemical reaction
Proteins are made on polyribosomes (polysomes).
- takes from 50 sec to 1-2 mins for a single ribosome to translate a protein
- output can be greatly increased if another ribosome hops on the mRNA and starts translating the same message as soon as the first one is out of the way
- common in both bacteria and eukaryotes
tetracycline
blocks binding of aminoacyl-tRNA to a A site of ribosome
streptomycin
prevents the transition from initiation complex to chain elongation, causing miscoding
chloramphenicol
blocks the peptidyl transferase reaction on ribosomes
cycloheximide
blocks the translocation step in translation
rifamycin
blocks initiation of transcription by binding to and inhibiting RNA polymerase
