translation and the genetic code Flashcards
the purpose of translation
to decode the mRNA and make the functional protein product of the gene
why does mRNA last longer in eukaryotes
- translation occurs in the cytoplasm but transcription occurs in the nucleus
- in prokaryotes both happen in the cytoplasm
“one gene, one collinear polypeptide”
the sequence of base pair triplets in the coding region of a gene specify a collinear sequence of amino acids in its polypeptide product
structure and function of proteins
- made of polypeptides which are long chains of amino acids
- amino acids have a free amino group, free carboxyl group and an R group (decides chemical nature)
peptide bonds
- join amino acids
- the carboxyl group of one AA is covalently attached to the amino group of another AA
4 levels of organization in proteins
primary: linear arrangement of amino acids
secondary: determined by the spatial organization of amino acids
tertiary: overall folding of the complete polypeptide
quaternary: only in some proteins, more than one polypeptide interact to make a functional protein
how many nucleotides are required to specify a single amino acid?
3 - triplet codon
- there are 64 possible codons, so some amino acids are specified by more than one codon
discovering the genetic code through homopolymers
- used an artificial mRNA containing only one repeating base pair
- the polypeptide contains a single amino acid
discovering the genetic code through mixed mRNAs
- used random copolymers
- there are 3 possible reading frames in the synthetic mRNA
- a ribosome that starts at a different frame will read the code for different amino acids
with the use of tRNAs what was determined between matches of codons and amino acids
short mRNAs of known sequence stimulated the binding of ribosomes and corresponding amino acid bound tRNA
- assisted in the identification of amino acids
degeneracy in the genetic code
- there are 64 codons and only 20 amino acids
- has to due with the wobble position
Wobble position
- there is flexibility in binding at the 3rd codon position (1st anti-codon position)
- this codon can be changed and still produce the same amino acid
anticodon
Crick’s wobble hypothesis
to account for degeneracy, tRNAs must exist for certain amino acids, and some tRNAs must recognize more than one codon
codon recognition and the wobble hypothesis
- stringent base pairing between the codon in mRNA and the anti-codon only occurs for the first 2 bases of the codon
- ## antiparallel base pairing happens between the anti-codon in the tRNA and the codon in the mRNA
the macromolecules of translation
- ribosomes, made up of polypeptides
- amino-acid activating enzymes
- tRNA molecules
- soluble proteins
ribosomes
- composed of proteins and several different rRNAs
- composed of 2 subunits - a large and small
- “RNA machine” with key roles in protein synthesis, including the formation of peptide bonds between amino acids
the role of tRNAs in translation
- the adapters between amino acids and the codons in mRNA
- anticodon of tRNA pairs with the codon of mRNA
- the AA is covalently attached at the 3’ end of the tRNA
- the normal ribonucleotides in tRNAs are often modified post-transcriptionally by enzymes
when do amino acids attach to tRNA
- tRNA is only attached to amino acids when you need to do translation
- you need energy (ATP) to attach the amino acid to tRNA
- the longer an amino acid is attached to a tRNA, the greater chance of degradation
activation of tRNA by aminoacyl tRNA synthetase
- cells contain at least one tRNA synthetase per AA
- the amino acid reacts with ATP producing AMP and PPi
- the amino acid is transferred to the appropriate tRNA and AMP is released
steps in translation (protein synthesis)
- initiation: transitional complex forms and tRNA brings the first AA to bind to start codon on mRNA
- elongation: tRNAs bring AAs one by one to add to polypeptide chain
- termination: release factor recognizes stop codon, translational complex dissociates and polypeptide is released
initiation in translation - prokaryotes
- IF3 binds to the small subunit and attaches it to the mRNA
- a tRNA charged with N-formylmethionine forms a complex with IF2 and GTP
- binds to the initiation codon while F1 joins the small subunit
- all initiation factors dissociate and GTP becomes GDP
- large subunit then joins initiation complex
initiation factors in translation - prokaryotes
IF-3: required to inhibit large (50s) subunit form binding (30s) small subunit
IF-2 and IF-1: position the fMet-tRNA to the ribosomal P site
- IF-2 GTP hydrolysis signals complex is ready for initiation by binding 2 subunits together
initiation complex in translation - prokaryotes
consists of large and small ribosomal subunits, initiation factors 1-3, and GTP
sites at the ribosome
A: aminoacyl site - site for incoming tRNA
P: peptide site - where you start protein synthesis
E: exit site - for ejecting tRNA
- fMet(or just Met is eukaryotes) is always the first AA to get incorporated and occupies the P site
the shine-dalgarno sequence
tells the ribosomes where to bind on mRNA in prokaryotes
16S rRNA
- component of the 30s small ribosomal subunit and contains the sequence complementary to the shine-Dalgarno sequence in the mRNA
- pairing between these 2 sequences positions ribosome near the AUG start codon
how does translation initiation differ in eukaryotes
- the first methionine is just Met not fMet
- no shine-dalgarno sequence
- ribosome initiation complex binds the 5’7-MG cap of the mRNA
- the Kodak sequence influences the efficiency of which AUG is used to start translation
- the polyA tail interacts with the 5’cap via cap-binding protein complex to promote initiation
kozak sequence
- translation start point in eukaryotes
5’GCC(A or G)CCAUGG3’ - the AUG of this sequence is the start codon
Elongation in translation
- tRNA binds to the A site of the ribosome
- the AA is transferred from the tRNA in the P site to the tRNA in the A site by the formation of a peptide bond
- ribosome moves along mRNA to position the next codon in the A site, results in the polypeptide tRNA being moved from A to P
- the uncharged tRNA is translocated from P to E and is removed
- a new tRNA binds in the A site and the cycle is repeated
translation termination
- occurs when a chain-termination codon enters the A site of the ribosome
- when a stop codon is reached a release factor (RF) binds to the A site
- RF1 or RF2 binding alters enzyme activity resulting in an H2O being added instead of an AA being added to the carboxyl terminus
- the binding of RF3 and GTP to the ribosome assists in dismantling the complex
release factors
- triggers translation termination
- attach a water molecule instead of incoming amino acid
- RF1 recognizes UAG and UAA stop codons
-RF2 recognizes UAA and UGA stop codons
-RF3 assists in dismantling the entire complex
quality control of RNA and protein
mRNA is eliminated that…
- has nonsense mutations
- which a stalled ribosome cannot complete proper translation]
- damaged by chemicals
- have unusual secondary structures
- molecular chaperones assist the proper folding of newly-synthesized proteins and are associated with the ribosome during translation
- the AAs of many proteins are modified post-translationally, can modify protein function and activity
