The NH₂‐terminal cleavage of Escherichia coli translational initiation factor IF3

Abstract

A short form of Escherichia coli translational initiation factor IF3, repeatedly found both in vivo and in vitro, lacking the positively charged N-terminal hexapeptide has been produced by mild trypsinization. The properties of this short form of IF3 have been studied. Compared to the long native form of the factor, the shortened IF3 displays a markedly decreased thermal stability and affinity for the 30 S ribosomal subunit, as well as a reduced biological activity in protein synthesis. Following the loss of the N-terminal hexapeptide, a second peptide bond (Lys-90-Val-91) becomes easily accessible to proteolytic attack suggesting that formation of the short IF3 may be the first step in the physiological degradation of the factor.

Initiation of mRNA translation in bacteria: structural and dynamic aspects

“…A 40-year-old model purporting a mutual incompatibility of IF3 and fMet-tRNA on the 30S subunit [ 176 ] has recently been reproposed [ 151 ]. However, as it was demonstrated that the claimed IF3/fMet-tRNA incompatibility was caused by an artefact due to the use of an N-terminally truncated factor [ 177 , 178 ], also the more recent revival of the vintage model turned out to stem from the use of an mRNA containing a too extended SD sequence regarded as non-canonical by IF3 [ 23 ]. Instead, FRET signals between fMet-tRNA (donor) and IF3 (acceptor) used as observables in kinetic analyses demonstrated the simultaneous presence of both ligands on the same 30S subunit and clearly showed that IF3 is dissociated during the 30S IC → 70S IC transition [ 23 ].…”

Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy

“…Model of the complex of initiation factor IF3 with the 30S ribosomal subunit+ A: Overview of the N-and C-domains of IF3 docked to the 16S rRNA in the 30S subunit seen from the solvent side+ B: Close-up of A displaying only the relevant features of the model+ C, D: A Ϫ908 and a ϩ908 rotation around the vertical axis of the image presented in B+ In all panels the 3D model of the 16S rRNA within the 30S ribosomal subunit is that based on EM reconstructions as described in Mueller & Brimacombe (1997) and based on the coordinates provided by the same authors specifically+ The 16S rRNA is shown (in its entirety only in A) completely in black but for the portions to which IF3 has been chemically crosslinked, namely, helices 45 (upper) and 25-26 (lower), which are shown in white+ Other nucleotides indicated are those protected by IF3 from kethoxal (yellow) and CMCT (orange)+ Nucleotide G791, which is partially protected from kethoxal and functionally implicated in IF3 binding by mutagenesis, is indicated in purple+ Nucleotides displaying hyperreactivity in the presence of IF3 to DMS (green) and kethoxal (turquoise) or hypersensitivity to RNase V1 (red) are also indicated+ Further details are found in the text (see Gualerzi & Pon (1990) for a review of these data)+ In all panels a P-site bound tRNA is displayed in magenta in the position indicated by Mueller & Brimacombe (1997)+ IF3 is represented as a blue tube in which the C-terminus of the N-domain and the N-terminus of the C-domain are displayed in darker blue and the residues affected by 30S interaction in yellow (NMR data) or red (mutagenesis or chemical modification data)+ activity of IF3 (Lammi et al+, 1987)+ Furthermore, Lys2 and Lys5, which were exposed to modification with pyridoxal phosphate in the absence of 30S ribosomal subunit, became protected in its presence (Ohsawa & Gualerzi, 1981)+ Because the modification of these lysines did not result in IF3 inactivation, however, Arg6 remains the most likely candidate for the establishment of an interaction with the ribosome+ The results of other chemical modifications and mutagenesis also allowed the identification of Cys65, Tyr70, Tyr75, and Lys79 as functionally important, yet perhaps only marginally (or indirectly) involved in binding to the 30S subunits+ The -SH group of Cys65 was modified by various reagents, including fluorescent-and spin-labels+ These experiments indicated that Cys65 is not essential for the biological activity of IF3 but that its rate of modification is slower in 30S-bound IF3 and a nitroxide spin-label at Cys65 became immobilized in titrations with 30S subunits, but not with nucleic acids such as random poly(A,U,G)+ It was suggested that Cys65, which is exposed in free IF3, is located at the edge of the IF3 binding site in the 30S-IF3 complex (Pon et al+ 1982a)+ Like Tyr107, which is located in the "primary RNA binding site" of the molecule (see above), both Tyr residues present in the N-domain (positions 70 and 75) were found to be accessible to lactperoxidase-catalyzed iodination in free IF3 and fully protected in 30S-bound IF3+ Isolated RNA, on the other hand, was found to protect Tyr70 from modification, but not Tyr75+ Furthermore iodination of Tyr70 did not prevent the binding of IF3 to the ribosome, but resulted in the formation of a partially inactive complex, whereas modification of Tyr75 did not produce any detectable inactivation of IF3 in vitro (Bruhns & Gualerzi, 1980)+ A direct influence of Tyr75 on the activity of IF3 in vivo is cl...…”

Evidence for an Active Role of IF3mt in the Initiation of Translation in Mammalian Mitochondria

“…Its predicted location is shown in the model (Figure 2C). This region was selected for mutagenesis since the first six residues of E. coli IF3 are important for the function of this factor (30). IF3 mt :2 is also located in the N-terminal domain in a loop between two β-strands facing away from the linker region.…”