Download (Protein Synthesis) Steps Initiation Elongation Termination

Prokaryote Translation (Protein Synthesis) Steps
Initiation
There are three initiation factors that along with GTP assist the mRNA in attaching to
the small 30S ribosomal subunit, and the initiator tRNA-formylMethionine complex to
bind to the mRNA. The Shine-Dalgarno sequence on the 5' end of the mRNA matches
up to the anti-Shine-Dalgarno sequence on the 3' end of the 16S rRNA of the small
subunit. Once the anticodon of the initiator tRNA-formylMet complex binds the AUG
start codon, the large 50S ribosomal subunit binds to the ribosomal binding site on the
mRNA and to a domain on the tRNA in the P (peptidyl) site. The A (aminoacyl) site is
empty as is the E (exit) site.
Elongation
The EF-Tu (elongation factor Tu) binds GTP and then the appropriate tRNA-AA at the
TΨCG loop and attaches it to the A site on the large ribosomal subunit. If the tRNA-AA
anticodon matches the codon, the GTPase domain on EF-Tu catalyzes GTP to GDP and
releases from the tRNA-AA. Now the amine group of this tRNA-AA complex can form a
peptide bond with the formyl-methionine amino acid in the P site. The peptidyl
transferase activity is controlled by the 23S rRNA and L2 and L3 proteins. At this point
the initiator tRNA is separated from its formyl-methionine amino acid. Another
complex EF-G with GTP binds where the EF-Tu-GDP complex dissociated from the
tRNA-AA in the A site and with the catalysis of GTP to GDP moves the ribosome three
nucleotides down the mRNA. This pushes the former A-site tRNA with the two amino
acid peptide fragment into the P site and opens the A-site once more. The unloaded
initiator tRNA moves into the E site and diffuses back into the cytoplasm to be reloaded
by its appropriate aminoacyl-tRNA synthetase. The EF-Tu-GDP complex is recharged
with GTP (or the high energy phosphate group?) exchanged from EF-Ts-GTP as it
becomes EF-Ts-GDP. It binds another tRNA-AA complex and brings it to the newly
opened A site. If the anticodon matches, then the process proceeds as above. This
continues until a stop codon is reached.
Termination
There are two RF (release factors) that recognize stop codons. RF1 recognizes UAA and
UAG and RF2 recognizes UAA and UGA. These bind the open stop codons assisted by
RF3 which is a GTP-binding protein that facilitates the binding of either RF1 or RF2 to
the large ribosomal subunit. At one end of RF1 or RF2 there is a conserved gly-gly-gln
amino acid sequence that is positioned near the P site tRNA-polypeptide complex. This
cleaves the ester bond between the polypeptide and the tRNA freeing both the
polypeptide and the tRNA and causing the ribosomal subunits to separate and fall off of
the mRNA.
Eukaryote Translation (Protein Synthesis) Comparison to
Prokaryote Steps
Initiation
There are six initiation steps with 8 or more protein factors that bind the initiator tRNAMethionine complex to the small 40S ribosomal subunit (43S), followed by binding this
new complex to the mRNA (48S), which involves scanning the Kozak sequence, after
which this complex is bound to the large 60S ribosomal subunit (80S). eIF2 assists the
initiator tRNA-Met complex (methionine is not formylated) to bind to small ribosomal
subunit with eIF2B to reactivate eIF2 by replacing its GDP with GTP. The 5' 7-methyl
guanosine cap is recognized by eIF4 (eukaryote Initiation Factor 4) and functions much
like the Shine-Dalgarno sequence in prokaryotes to help position mRNA on the initiator
tRNA-Met-40S ribosomal subunit for proper translation. Scanning occurs for the Kozak
sequence [CC]RCCAUGG with R coding for A or G with A being more efficient to
initiate translation at the start codon within the Kozak sequence. eIF5 helps the large
60S ribosomal subunit bind to this complex. [eIF6 has previously prevented precocious
binding to the complex before the critical steps were completed. If a 3' polyA tail is
present on the mRNA, this will fold back over the initiation complex binding to the 5'
7mG cap binding proteins.
Elongation
The eukaryote elongation steps are very similar to those in the prokaryotes. The EF-GGTP complex is EF2-GTP in eukaryotes. The protein elongation factor 1 alpha (EF-1α)
is a key GTP-binding enzyme in protein synthesis which carries out the same function as
the prokaryote EF-Tu. [Small groups of organisms in all eukaryote kingdoms use
elongation factor-like protein (EFL) for the same purpose.] It brings the tRNA-AA
complex to the large ribosome subunit Aminoacyl (A) site for the tRNA anticodon to
pair with the mRNA codon. Once pairing of complementary bases occurs, the EF-1α
GTPase domain catalyzes GTP to GDP and EF-1α releases the tRNA-AA so that the
amino acid can now form a peptide bond with the amino acid in the Peptidyl (P) site.]
Termination
The eukaryote release factor eRF1 recognizes all three stop codons: UAA, UAG and
UGA. Although it is structurally different from prokaryote RF1 and RF2 with one
domain mimicking a tRNA with its anticodon, it has the conserved gly-gly-gln sequence
needed to cleave the ester bond holding the polypeptide to the last tRNA in the P site. A
second release factor eRF3 functions much like prokaryote RF3 using GTP to help eRF1
release the polypeptide and to cause the ribosomal subunits to dissociate and fall away
from the mRNA.