Release of Polypeptide Chain Initiation Factor IF-2 During Initiation Complex Formation

Abstract

Polypeptide chain initiation factor IF-2 binds to 30S ribosomal subunits. This binding is enhanced by IF-2, one of the bacterial polypeptide chain initiation factors, is essential for the binding of both GTP and fMet-tRNA in a 30S initiation complex (1). This complex contains a 30S ribosomal subunit, mRNA, fMet-tRNA, and GTP (2,3). The combination of a 50S-subunit with the 30S complex results in the hydrolysis of GTP to GDP and Pi, concomitant with the formation of a functional 70S initiation complex (2,3). It has become clear that IF-2 is responsible for hydrolysis of GTP in the initiation process. In fact, under conditions uncoupled from initiation, IF-2 catalyzes the hydrolysis of GTP to GDP and Pi in the presence of both ribosomal subunits (4, 6). This activity can be coupled to initiation complex formation (6). Hence, IF-2 participates in the formation of a 30S initiation complex, as well as the subsequent transformation of this species into a 70S initiation complex.Recent studies have demonstrated that IF-2 functions stoichiometrically during 30S initiation complex formation, but catalytically during 70S initiation complex formation (2, 7), i.e., in the presence of both 30S and 50S subunits. Hence, it would appear likely that IF-2 binds to a 30S subunit and is released upon addition of a 50S ribosomal subunit. The released factor would then be able to catalyze another round of initiation. Examination of crude cell extracts reveals that IF-2 activity is localized on 30S subunits (8), and that no IF-2 (nor other initiation factors) are found on 70S ribosomes.In this communication, we provide a direct demonstration that IF-2 binds to 30S subunits. Upon formation of a 70S initiation complex, IF-2 is released from the ribosomes. In contrast to IF-1 and IF-3, release of IF-2 requires the hydrolysis of GTP. MATERIALS AND METHODSInitiation factors were purified (9) from Escherichia coli MRE-600. IF-1 and IF-3 were homogeneous, while IF-2 was about 90% pure by the criteria of native and Na dodecyl S04 disc-gel electrophoresis. Salt-washed ribosomes were prepared from E. coli Q13. Such ribosomes exist primarily as dissociated subunits. The preparation of f [3H]Met-tRNA, as well as the source of all reagents, has been described (9).Assay of fMet-tRNA and IF-2 Binding to Ribosomes. Reaction mixtures (0.125 ml) contained 50 mM Tris HCl (pH 7.8), 80 mM NH4CI, 10 mM magnesium acetate, and 4 mM 2-mercaptoethanol. In addition, the complete system con- (w/v) sucrose gradient containing reaction buffer, and centrifuged for 75 min at 55,000 rpm at 40 in a Spinco SW65 rotor. 0.2-ml fractions were collected and radioactivity was determined by counting aliquots in Bray's counting solution. The location of ribosomes was determined by monitoring the absorbancy at 260 nm. Gradient fractions were assayed for IF-2 activity by addition of 50-,ul aliquots to the complete reaction mixture described above, from which IF-2 had been omitted.

Initiation of Protein Synthesis in Bacteria

“…However, B. stearothermophilus IF2 can hydrolyze GTP in the absence of ribosomes when 20% ethanol is included in the reaction mixture (64). GTP hydrolysis in translation initiation has been suggested to be important for the release of IF2 from the 70S initiation complex (108,114), and for the adjustment of initiator tRNA in the ribosomal P-site (103). The crucial importance of GTP hydrolysis in translation initiation and its direct relation to cell viability have been confirmed by several studies of G-domain mutants of IF2 (98a, 100, 106, 115).…”

Thiostrepton Inhibition of Initiation Factor 1 Activity in Polypeptide Chain Initiation in Escherichia coli

“…The hydrolysis of GTP coupled to fMet-tRNA binding is also markedly inhibited (4). In initiation reaction mixtures containing limiting levels of IF-2 with respect to ribosomes, maximum fMet-tRNA binding requires recycling of the factor (18,19,21). In contrast, when IF-2 is present in molar excess with respect to ribosomes, the catalytic role of IF-2 is unnecessary.…”

“…IF-2 is released from the initiation complex and can recycle to catalyze another round of initiation (18)(19)(20).…”

Effects of Antibiotics, N‐Acetylaminoacyl‐tRNA and Other Agents on the Elongation‐Factor‐Tu Dependent and Ribosome‐Dependent GTP Hydrolysis Promoted by 2′(3′)‐O‐l‐Phenylalanyladenosine

“…AcPhe-tRNAPhe and AcTyr-tRNATYr preparations also contained 35 and 30 x, respectively, of deacylated tRNAPhe and tRNATy'. However, the effects described cannot be attributed to these contaminants, since control experiments showed that the presence of 80 pmol deacylated tRNAPhe in the reaction mixture caused only a 12 "/o inhibition of AdoPhe-dependent GTPase and that lower concentrations [6], RNA phage replication [24] and possibly in the regulation of transcription [25]. Multifunctional proteins may contain autonomous structural regions (domains) that carry out the different functions [26].…”

“…Howcver, at the molecular level very little is known of the interactions that trigger G T P hydrolysis. In the casc of elongation factor (EF) Tu, the reaction normally requires ribosomes, mRNA and aminoacyl-tRNA and is coupled to the binding to the ribosome of the aminoacyltRNA specified by the mRNA ( [ 6 ] , review). Our recent work, however, has shown that G T P hydrolysis can occur when only EF-Tu, ribosomes and 2'( 3')-O-~-phenylalanyladenosine (AdoPhe), i.e.…”