Translation and protein synthesis Flashcards
Components of translation
Amino acids: All amino acids in a protein have to be present at the time if protein synthesis (essential AA)
tRNAs: adaptor molecules, at least one tRNA per AA, two important sites on tRNA (AA attachment site and the anticodon sequence), they have highly ordered structure
mRNAs: translated in the 5’->3’ direction (in bacteria, transcription and translation are coupled) (regulation of protein expression is largely controlled at the transcriptional level
aa-tRNA synthetase: family of enzymes that attach the AA to the corresponding tRNAs
ribosomes: ribonucleoprotein complex, (prok= 70s (30 and 50)) (euk= 80 (60 and 40))
initiation, elongation and termination factors
ATP and GTP energy sources: cleavage of 4 high energy bonds are required for the addition of one AA, initiation and termination require ATP and GTP
Ribosomal composition
Bacterial Ribosome (70s = 30s + 50s) 50 s= 55, 23, 36 proteins 30s= 16
Eukaryotic Ribosome (80s= 60s + 40s) 60s= 5s, 28, 5.8, 40s= 18s
The two subunits have mRNA running in between
Chloroplasts and mitochondria have ribosomes like bacteria
Genetic code
20 common genetically coded amino acids
four letter code in groups of two is insufficient because it would only code for 16
four letter code in groups of three is sufficient (64 codes)
64 codons (61 codons for amino acids)
Code has the following properties:
- its specific (a specific codon ALWAYS codes for the same AA)
- its universal (mito is different)
- its redundant/degenerate (a given AA may have more than one codon (mostly in the third position)
- its non overlapping and comma less( code is read from a fixed starting point and is continuous three at a time. Frameshift mutations occur if the reading frame is altered
Third base is less important in binding to tRNA, first codon establishes the reading frame
Temination codons: UAA, UAG, UGA
AUG is the intiation codon (also codes for Met)
Codon properties
- termination codons dont code for an amino acid
- Altered codons:
silent mutations- when the codon base is changed to a codon that codes for the same AA
missense mutation- codon base is changed to codon that codes for a different AA
nonsense mutation- codon changed to a termination codon
degeneracy of genetic codes
Most amino acids have more than one codon
only Met and Trp have one codon
some codons are less subject to causing a mutation in an amino acid because highly degenerate/redundant
Genetic code is resistant to mutations b/c of degeneracy, some mutation can still code for the same AA (silent mutation)
when the codon is mutated in the first base it usually produces a missense mutation
codon recognition by tRNA
tRNA anticodons recognize codons
binding follows rules of complementary base pairing
mRNA is read 5’->3’ by a flipped (3->5) anticodon
Molecular recognition of codons in mRNA by tRNA
tRNA anticodons recognize codons (complementary, hydrogen bonding, antiparallel)
wobble hypothesis (tRNAs can recognize more than one codon for a specific amino acid due to a wobble at the 5’’ end of the tRNA anticodon, forming non traditional base pairs, result is that there does not need to be 61 different tRNAs to read the 61 different codons)
suppressor tRNAs (non sense suppressors are tRNAs whose anticodons have been mutated to where they incorporate an amino acid at the termination codon) (they supress normal termination of a protein)
Stages of protein synthesis
- tRNA is activated via aminoacylation
- Initiation (mRNA and amino acylated tRNA bind to the small subunit of ribosome, then the large subunit sandwiches the mRNA/acylated tRNA)
- Elongation: ribosome moves along the mRNA, matching tRNAs to each codon and catalyzing peptide bond formation
- termination: translation stops at a stop codon, the mRNA and protein dissociate, ribosome is recycled
- protein fold into active conformation
Activation of Amino acids
Amino Acid + tRNA + ATP -> aminoacyl-tRNA+ AMP + PPi
Via: aminoacyl tRNA synthetase and Mg 2+
Aminoacyl tRNA synthetases:
Each enzyme binds a specific amino acid and the matching tRNA,
most cells contain 20 different aa-tRNA synthetases, one for each amino acids
The aa-tRNA synthetase needs to be specific for both the AA and the tRNA (anticodon)- confers the binding specificity
matching the tRNA and the amino acid is like a second genetic code via molecular recognition
initiation
A processed (poly- pro, or mono cistronic for euk) mRNA is processed,
in prok, the 16 s rRNA of ribosome is involved in the correct positioning of at the translation start site (AUG- augment)
The alignment of the small subunit on the AUG start codon is dependent on the upstream Shine Dalgarno sequences (that base pairs with the 3’ end of the small subunit)
Initaition codon: recognized by special initiatior tRNA: facilitated by IF2 in prokaryotes and eIF-2 in eukaryotes
in prokaryotes and mitochondria : the Met start amino acids starts with an N-formyl Met (removed before termination)
in eukaryotes: its just Met
fMet or Met is the first amino acid in a peptide (coded by AUG), all organisms have two tRNAs for Met( in prok/mito there is a tRNA for Met and a tRNA for fMet)
Eukaryotes begin with regular Met but still have a special tRNA
IF3 or eIF 3 keeps the small and large ribosomal subunits apart and brings in the mRNA on to the P site of the small subunit (IF 1 blocks the A site)
IF2 brings in the initation Met (with initiator tRNA)
IF 1, 2, and 3 are removed with the hydrolysis of GTP, so the subunits come together
Elongation
dependent on A site entry
An incoming amino acyl tRNA is delivered by EF-Tu upon GTP hydrolysis (EF-Tu is a GTPase and its activity sets the rate of protein synthesis)
Peptidyl transferase catalyzes the peptide bond formation so that the Amino acid from site P is popped on the the A site
The empty tRNA (deacylated tRNA) is bumped into the E site via EF-G translocase and GTP hydrolysis
The double AA on the tRNA then goes to the P site and the awaits the next A site
Termination
Release factor recognize stop codons at the A site, they change the specificity of peptidyl transferase to recognize water instead of an amino group-> leads to peptide release
eukaryotes vs prokaryoted in protein synthesis
Ribosomes: different sizes
Initiator tRNA:
in euk->met- tRNA i
in prok-> fmet- tRNAi
Initiation of translation: selection of AUG in prok depends on the shine delgarno sequence that base pairs with 16s subunit
but in eukaryotes, the 5’ AUG in mrna is the initiator Met, the 40 s loads on mRNA 5’ end and scans for AUG (needs ATP and helicase) eIFs all come together and initiate it, then once elongation begins all the EIFs fall off
48 S complex (eIFs and the small subunit) unwinds any helix and scans to find the first AUG, (eIF4 has the helicase activity)
Once AUG is found, 60 s come in and 80s is formed and release of eIF (initiation complex is formed)
Translational control by phosphorylation
Phosphorylation of EIF4E needs to be phosphorylated to find the AUG and unwind
Factors that stimulaate translation of EIF4e proteins (TNF a, insulin, growth factors, IL1, p21 ras)
Phosphorylation of EIF2 blocks formation of 43 S preinitialion complex to inhibit translation
Diptheria toxin blocks euk translation by inhibiting tanslocation via ADP ribosylation of EF2 translocase
