569 research outputs found
Ribosome Rescue and Translation Termination at Non-Standard Stop Codons by ICT1 in Mammalian Mitochondria
Release factors (RFs) govern the termination phase of protein synthesis. Human
mitochondria harbor four different members of the class 1 RF family:
RF1Lmt/mtRF1a, RF1mt, C12orf65 and ICT1. The homolog of the essential ICT1
factor is widely distributed in bacteria and organelles and has the peculiar
feature in human mitochondria to be part of the ribosome as a ribosomal
protein of the large subunit. The factor has been suggested to rescue stalled
ribosomes in a codon-independent manner. The mechanism of action of this
factor was obscure and is addressed here. Using a homologous mitochondria
system of purified components, we demonstrate that the integrated ICT1 has no
rescue activity. Rather, purified ICT1 binds stoichiometrically to
mitochondrial ribosomes in addition to the integrated copy and functions as a
general rescue factor, i.e. it releases the polypeptide from the peptidyl tRNA
from ribosomes stalled at the end or in the middle of an mRNA or even from
non-programmed ribosomes. The data suggest that the unusual termination at a
sense codon (AGA/G) of the oxidative-phosphorylation enzymes CO1 and ND6 is
also performed by ICT1 challenging a previous model, according to which
RF1Lmt/mtRF1a is responsible for the translation termination at non-standard
stop codons. We also demonstrate by mutational analyses that the unique
insertion sequence present in the N-terminal domain of ICT1 is essential for
peptide release rather than for ribosome binding. The function of RF1mt,
another member of the class1 RFs in mammalian mitochondria, was also examined
and is discussed
Identification of Distillation Process Dynamics Comparing Process Knowledge and Black Box Based Approaches
Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu
The presence or absence of deacylated tRNA at the E site sharply influences the activation energy required for binding of a ternary complex to the ribosomal A site indicating the different conformations that the E-tRNA imparts on the ribosome. Here we address two questions: (i) whether or not peptidyltransferase—the essential catalytic activity of the large ribosomal subunit—also depends on the occupancy state of the E site and (ii) at what stage the E-tRNA is released during an elongation cycle. Kinetics of the puromycin reaction on various functional states of the ribosome indicate that the A-site substrate of the peptidyltransferase center, puromycin, requires the same activation energy for peptide-bond formation under all conditions tested. We further demonstrate that deacylated tRNA is released from the E site by binding a ternary complex aminoacyl-tRNA•EF-Tu•GDPNP to the A site. This observation indicates that the E-tRNA is released after the decoding step but before both GTP hydrolysis by EF-Tu and accommodation of the A-tRNA. Collectively these results reveal that the reciprocal linkage between the E and A sites affects the decoding center on the 30S subunit, but does not influence the rate of peptide-bond formation at the active center of the 50S subunit
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