Biochimie ( 1991 ) 73, 1557-1566

15 57

O Socirt6 franqaise de biochimie et biologie molrculaire / Elsevier, Paris

Structural and functional domains

o f E coil i n i t i a t i o n f a c t o r I F 2

S Laalami l, C Sacerdot ~, G Vachon 2, K Mortensen 3, HU Sperling-Petersen 3, Y Cenatiempo 2, M Grunberg-Manago~* Ilnstitut de Biologie Physico-Chimique, 13, rue Pierre et Marie Curie, 75005 Paris; 2Labm'atoh'e de Biologie Mold~'ulaire. URA CNRS 1172, Universit~ de Poitiers, 40, ~,'enue du Recteur Pineau, 86022 Poitiers Cede.r. France: 3Laboram13. ~ Blodesign. Department of Chemistry, Aarhus University, DK-8000 Aarhus, Denmark

(Received 9 October 1991; accepted 4 November 1991 )

Summary --Initiation of translation in prokaryotes requires the participation of at least three soluble proteins: the initiation factors IF1, IF2 and IF3. Initiation factor 2, which is one of the largest proteins involved in translation (97.3 kDa) has been shown to stimulate in vitro the binding of fMet-tRNAr Met to the 30S ribosomal subunit. After formation of 70S translation initiation complex, IF2 is believed to participate in GTP hydrolysis, thereby promoting its own release. Here we review evidence which indicates the functional importance of the different structural domains of IF2, emphasizing new information obtained by in rivo experiments. translation initiation factor 2 / IF2~ and IF2[~ / G bindin~ protein / G domain

Introduction

The first step o f protein synthesis in prokaryotes requires at least three proteins called initiation factors IF1, IF2 and IF3 [1]. The existence of temperature-sensitive mutants in IF2 and IF3 genes indicates that they are essential genes [2, 31. However, the role o f initiation factors in vivo is still not well documented. Of~ the three factors, the role of IF2 is best understood. It promotes the formation o f the translation initiation complex by bringing the initiator fMet-tRNAr Met to the ribosomal 30S subtmit [ 1]. The infB gene encoding IF2 is located on the E coli c h r o m o s o m e at 69 min [4] and is part o f a complex multifunctional operon comprising the genes metY, p l 5 A , nusA, infB, p l 5 B and p35 [5-7]. These genes encode a minor form o f initiator tRNA, a protein involved in transcription termination and three proteins o f unknown function, identified as p l 5 A , p l 5 B and p35 with molecular weights of 15 and 35 kDa. Striking similarities in internal i;~B operon structure have been found between E coli (Gram-) and B subtilis (Gram +) [81, although the chromosomal location o f the two operons is dissimilar. The fact that the organization o f the operon has been preserved in the two organisms which have been separated for more than one billion years o f evolution argues that *Correspondence and reprints

the organization should reveal conserved and unique aspects o f translational control strategies. Sequence analysis of the il~B gene showed that near the center there is a region characteristic o f proteins which bind GTP. This motif was originally identified in small mammalian proteins o f the ras oncogene family p21. It is related to yeast RAS proteins and has been called the G-domain. It has also been identified in various signal transducing membrane proteins 191. Therefore, IF2 belongs to the class o f the GTP-binding proteins [9]. In E coli, four proteins in addition to IF2 exhibit strong homology to the G domain consensus: they are translational elongation factor Tu (43 kDa), translational elongation factor G (76 kDa), LepA (66 kDa) and Era (35 kDa) [10--!4]. LepA is involved in secretion, while the role in vivo of Era has not yet been clarified. Like IF2, EFTu and EF-G are well characterized GTP-hydrolyzing proteins belonging to the translational apparatus. It is to be noted that all G-proteins including mammalian and yeast RAS are much smaller than IF2. The three proteins involved in translation, IF2, EF-Tu and EF-G all require one or both subunits o f the ribosome to trigger their GTPase activity. However, the cloning of a fragment o f EF-Tu corresponding to the G-domain has demonstrated its intrinsic GTPase activity 115]. It was suggested that the rest of the EF-Tu molecule is involved not only in aminoacyl tRNA binding and interaction with EF-Ts and ribosomes but also in regulating the activity of the G-domain localized in

1558

S Laalami et al

the N-terminal part of the molecule. Sequences of IF2 and Tu show that there are two regions in EF-Tu in the proximal and middle positions of the gene which display extensive homologies with middle portions of infB (base 1318-1384) and (1513-1644). Figure 1 shows the alignment of the DNA sequences and the corresponding amino acids [ 16] When one compares IF2 and EF-Tu, one wonders why IF2 is twice as large as EF-Tu. The roles of IF2 and EF-Tu are very similar. Both promote the binding of tRNA to the ribosome. Furthermore, the G-domain in the G proteins is situated in the N-terminal part of the molecule, while in IF2 it is localized in the central part. A related question is the in vivo function of the N-terminal part of IF2. in vivo IF2 is present in cell lysates in two forms, named ¢x and [l which differ in size: 97.3 kDa (¢x) and 79.7 kDa ([~) [1, 17]. During purification, a third form appears with a molecular weight of 65.4 kDa. Whereas the 65.4 kDa IF2 is a truncated form resulting from proteolysis during purification [16], IF2cx and IF2~ appear to coexist in the bacterial cell in a constant 2/1 molar ratio [ 1].

IF2 exists in two forms

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It was initially shown that the cz and 13forms of IF2 are related immunologically and exhibit common tryptic peptides. Amino acid sequence at the Nterminus of the two forms as well as the overall amino acid sequence deduced from the nucleotide sequence of the IF2 gene demonstrated that IF213 is precisely identical to IF2ct from the C-terminus, but lacks 157 amino acids from the N-terminus [18]. When subcloned as a 3-kbp segment, infB expresses both forms of IF2. S1 nuclease mapping experiments indicate that IF2tz and IF213 are translated from a single mRNA harboring two independent translational start sites situated in frame. N-terminal amino acid sequence of IF2o~ identified an AUG triplet as start site consistent with the size of IF2o~. This AUG triplet is preceded by a strong Shine-Dalgarno sequence. The N-terminal amino acid sequence for IF2~ indicates GUG as a start site. It is preceded by a purine-rich sequence with a weaker Shine-Dalgamo sequence, only three bases complementary to 16S rRNA 3' terminus. The first evidence that IF213 is expressed from an independent start site is based on in vivo synthesis of IF213-13-galactosidase hybrid protein after deletion of the upstream region including the Shine-Dalgamo sequence in front of the AUG of

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Fig 1. Sequence homologies between IF2 and EF-Tu. Homology between the G-domains (residues 391-540) and EF-Tu (residues 12-180) was determined by maximizing identical or near identical residues. Identical residues are in squared boxer and functionally conserved residues are in rounded boxes. Residues absent in the strands compared are shown by dashes. The sequences forming 13-strands or o~-helicesin the three-dimensional model (fig 2) are indicated (reprinted from [ 16]).

The structural and functional domains of E coli IF2 IF2~'s RBS [18]. Further evidence using different deletions of the upstream and downstream region of the IF21~ start codon unambiguously show that GUG located 471 bases downstream from the IF2t~ start site is indeed the codon where IF2~ synthesis is initiated principally [18, 19]. More recently, a close examination of IF213 synthesis on SDS-PAGE gels of lower polyacrylamide percentage from wild-type plasmids shows that IF213 is actually a double band [20]. The two species of IF2~ proteins (IF2131 and IF2132) have been separated by FPLC chromatography according to the procedure of Mortensen et al [21]. Their Nterminal sequences were determined and it was found that there is a second start site for IF21~ at an AUG (514-516) located 7 codons downstream of the GUG start codon (493--495) fer IF2I~I, permitting the synthesis of a protein missing 7 amino acids: IF21~2. Compared to the identified form of IF2~, IF2131 is the major species in normal physiological conditions as N-terminal sequencing of a wild-type IF2~-I]-galactosidase hybrid protein (which is expected to contain both IF2~l, and IF2~12 fusion forms) gives the IF2~ll sequence. In any case, the data also show that IF2131/132 can be synthesized even when IF2o~ formation is entirely prevented by eliminating all its initiation signals. This unambiguously eliminates the possibility that IF213 was a proteolysed product of IF2ct. It also eliminates a restart mechanism for synthesis of IF21~.

Structure of initiation factor IF2 Structural features of amino acid sequence

Evaluation of the amino acid sequence of IF2 reveals that the first region (residues 104--155) has a very abnormal amino acid composition consisting essentially of only five different amino acids (16 alanine, 16 glutamic acid, 5 lysine, 7 arginine, 7 glutamine). The strict repetition of alanine residues every four amino acids, the pattern Arg-Glu-Ala repeated six times and a periodicity of eight residues are quite remarkable. A helical secondary structure is predicted for this region [22] (fig 3). The location of alanine every four residues suggest that a four-fold helix (eg w helix) is a possibility. The regularity of alanine distribution and of charged groups maximizing neutralization is quite striking. The second region concerns residue 167-214. Its amino acid composition is also rich in alanine, glutamic acid, lysine and arginine. The pattern Glu-GluAla-Arg-Arg is repeated three times and a periodicity of eight residues is seen. A helical structure is predicted. In addition, the first and second region show amino acid (50%) and nucleotide (57%) sequence homologies. This part of the protein may alternate between flexible and helical conformations [22].

1559

A six-domain so'ucture model for IF2

A working hypothesis has been proposed for the structure of IF2 [23]. It is based on data from DNA sequence, peptide sequences and proteolysis pattern (P Riisgaard et al, unpublished data) and prediction of secondary structure. Furthermore, small-angle neutron scattering experiments on IF2o~ indicate a slightly elongated overall shape approximately 900 × 900 x 960 nm as calculated from the radius of gyration [24]. The model for the 890 amino acid residue large IF2ct consists of six structural domains (domains I-VI). Since IF2t~ and IF213 are both active in vitro and in vivo, it is unlikely that the N-terminal part of IF2cc, amino acids 1-103, constitutes more than one structural domain-domain I. This N-terminal domain of IF2o~ is followed by the two regions containing internal sequence homologies described above (residues 104-214). They constitute a flexible hydrophilic link between domain I and domain II. The C-terminal limit of domain II can be defined by the very sensitive site of proteolysis that exists between Lys 289 and Arg 290. We have published a detailed structural model for the IF2 G-domain: amino acid 392 to 540 of IF2t~ (fig 2). In the six-domain model, this domain IV is structurally the best defined part, as discussed below. The 219 C-terminal amino acids of IF2t~ have been isolated by proteolytic cleavage between amino acids Lys 671 and Ser 672 (P Riisgaard et al, unpublished data). This fragment, which is extreme!y stable under non-denaturing conditions, forms domain VI (amino acids 672-890). The remaining parts of IF2tx: amino acids 290-391 and 541--671 are the flanking regions between domain II and IV and between domain IV and VI respectively. Considering their size of approximately 10 kDa, these polypeptide chains were suggested to form two distinct structural domains, domains IU and V, which show 35% amino acid sequence homology. This six-domain tertiary structural model of IF2 is considered as a working hypothesis for our ongoing genetic and er, zymatic modifications of IF2. A proteolysed form of IF2: IF2y During purification of IF2o~ and IF2~l, a smaller form, IF2),, is generated. It has an apparent mass of 70 kDa, as determined by SDS-polyacrylamide gel electrophoresis [16, 17]. Since its presence in fresh lysates is not detected, it was likely that IF2y is generated by partial proteolysis of native 1F2 proteins during purification. The sequence of the 15 N-terminal amino acids was determined and indicates that IF2y has arisen by cleavage of the Lys 289-A~"g 290 bond of IF2. The secondary structure prediction places the two

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Fig 3. Primary and second,~ry structures of IF2. A schematic representation of the primary structures of IF2~, IF2~ and IF2y is shown (reprinted from [ 16]).

Fig 2. Structural cartoon for the tertiary structure model of

IF2 G-domain. The arrows represent ~-szrands (I-VI), and curled ribbons represent o~-helices (AE). Arabic numbers indicate IF2ct amino acid residues at the ends of secondary structure elements. A GDP molecule is shown in black (reprinted from [ 16]). residues in a loop connecting an o~ helix and a 13 strand. It has been demonstrated that the cleavage is due to a membrane linked protease ornpT [25]. The Cterminal amino acid seqLlence of IF27 shows that it possesses the entire C terminus of IF2cx. Calculated from the sequence, IF2T has a molecular mass of 64.8 kDa. The relation between the primary structure of IF2~, IF21 and IF27 is shown in figure 3. The Ntermini of IF2cx, IF21~I (Val 158), IF2y (Arg 290) are indicated (the N-terminus of IF212 is Met 165). The secondary structure prediction is shown schematically for IF2a and in more details for the region (residues 392-540) which is the GTP binding domain. This region is a part of all three forms of IF2. The secondary structure contains six 1-strands numbered I-VI and four q-helices numbered A, B, C and E. The 0~helix D, which emerges from sequence comparison with EF-Tu (see fig 1), is not observed in this prediction. The two regions in IF2 that are homologous to the phosphoryl- and guanine-binding regions in EF-Tu are labeled in figure 4. A striking feature of the arrangement of the or-helices and p-strands is that the segments are found in the same order in IF2 and EFTu. This feature strongly suggests that the higher

order structures of the two factors are similar. Of the 150 residues of IF2 compared to those of EF-Tu, 59 are identical and another 24 are closely related. A computer model of the G-domain of IF2 was built on the basis of this sequence homology and the crystallographic model of E coli EF-Tn:GDP (fig 2). The overall folding of the IF2 peptide chain in the model is similar to, that of EF-Tu [10, 11, 26], an Ix/l~-structure with a curved parallel I~-sheet sandwiched between two layers of Q-helices ~,vlxclvo~n~i.w. The guanine-binding site is situated at the carboxyl end of the sheet, with the guanine moiety wedged in a hydrophobic pocket between 1-strands V and VI. The phosphates are situated at the N-terminus of the o~-helix A, giving a favorable interaction between the dipole moment of the t~-helix and the negative charge of the diphosphate group. Amino acid residues 398-403 form a loop connecting [~-strand I and t~-helix A, and residues 404-405 form the beginning of t~-helix A. This loop is responsible for the binding of the pyrophosphate group and exhibits the characteristic sequence GIy-X-X-X-X-Giy-Lys-Thr, where the lysine is directly involved in the binding of the 1-phosphate. The segment 498-501, with the sequence AsnLys-Ile-Asp, is responsible for guanine recognition by hydrogen bonding from Asn-498 to the keto group of the base and from Asp-501 to the amino group. C o n s t r u c t i o n o f a null m u t a t i o n in IF2

As an approach to examining the functional significance of IF2 in vivo, a strain of E co# was constructed where the chromosomal copy of the infB gene has been inactivated by insertion of an antibiotic cassette [27]. As inj~B was suspected to be an essential gene, a functional infB gene was supplied in trans from a thermosensitive lysogenic L phage integrated at att~.. The bacteria survive heat curing of the 7~ infB carrying phage, provided that a viable infB allele is supplied in trans.

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Expression of truncated forms of infB Different shortened derivatives of IF2 have been constructed by recombinant DNA techniques. l) A large in-frame deletion that removes the first 42.8 kDa fragment of IF2(~ and 25.2 kDa fragment of IF213 preceding the G-domain. This construct produces a 55 kDa protein (IF2Al) which consists of the 17 kDa G-domain and the entire C-terminal part of IF2 (fig 4). 2) Two other deletions were constructed removing 23.4 and 35 kDa from the C terminus of IF2Al producing proteins of 31.1(IF2A2) and 17.6 kDa (IF2A3) respectively. The latter contains only the region corresponding to the GTP-binding domain. These deletion constructs were cloned in an expression vector downstream of the tandem 2Lpromoters pR and pL but containing the translation initiation signals of IF20~ (Shine-Dalgarno sequence and natural in-

itiation triplet AUG remain intact). The deletion starts at the fourth codon of the infB gene. Maxicell analysis of the protein coded by these plasmids shows that all three constructs express proteins of the expected masses: 50 000, 31 000 and 17 000 [27]. The entire infB gene was cloned in the same vector to express both IF20~ and IF2~. The 55 000 M r protein was detected by Western blotting of total cell extracts of plasmid carrying strains and probing with anti-IF2 antibodies. The 31 000 M, protein was also detected by anti-IF2 antibodies, but the 17 000 M, protein was not recognized effectively. This is presumably due to a lack of major IF2 antigenic determinants. The plasmids were transferred into an IF2 conditionally deficient strain and tested for their ability to permit survival of the bacteria after heat curing of the ~, infB lysogenic phage. Survivors were detected at similar frequencies in strains expressing either the 55 000 M, polypeptide or the wild-type infB gene from plasmid encoded promoters. Analysis of Western blots of the cured strains confirmed the complete lack of wild-type IF20t and IF213 proteins (fig 5). No survivors were detected in strains containing the two other infB fragments expressing proteins of 31 000 M, or 17000 M r. The strains harbouring the plasmid expressing the 55 000 M r 1F2AI protein produced smaller colonies on agar plates, were of uniform size and remained stable throughout multiple purifications. On the other hand, cells expressing an IF2 protein deleted for the Cterminal quarter of amino acids are not viable, thus demonstrating that this part of the protein is essential.

s Laalami et a!

1562

When testing the influence of temperature on the gTowth rate of the various strains the following observations were made: IF2AI being expressed from kpL, kPR promotors which are efficient at high temperatures when the repressor is inactivated grew only at temperatures above 40°C with optimal growth at 42°C (doubling time 34 min). Strains expressing in1B constitutively from a plasmid encoded promotor grew well at all temperatures (doubling time 3033 min at 37°C). However, when wild-type IF2 was expressed from the tandem ~, promotors, optimal growth was achieved at 37°C (doubling time 29 rain). At 42°C, growth was severly retarded, suggesting that the excess amount of IF2 synthesized at 42°C is inhibitory to cell proliferation. Quantitative immunoblotting experiments showed that reasonable growth depending only on IF2A1 was observed when the truncated IF2 protein was at least in five-fold molar excess compared to wild-type IF2tx and ~ levels. In order to test complementation at 37°C, a plasmid deleted for the ~. repressor was constructed, thus exp~ssing the 55 000 M, IF2A1 protein constitutively at all temperatures from the unrepressed EpL-pR promoters. This plasmid allowed growth of the cured strain at 42°C and 37°C, even though at reduced rates when compared to cultures grown at 42°C in the presence of the repressor. The appreciable reduction m growth rate (from 34 to 73 min) was not due to a

1

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Fig 5. Western blots of the cured strains carrying different infB plasmid constructs. Lane 1: cured strain carrying a p!asmid with infB under control of pL, pR ~, promoters (grown at 37°C). Lane 2: cured strain carrying a plasmid with infBAl under control of pL, pR ~, promoters (grown at 42°C). Lane 3: strain SL598R carrying a null mutation in the chromosomal copy of infB and a functional copy of infB on a ~, phage (grown at 30°C). Lane 4: cured strain carrying a plasmid with a nusA-infB insert (grown at 37°C). Lane 5: same as lane 4 with a deletion in infB that eliminates IF2o~ expression. Lane 6 and 7: same as lane 4 with each one of the double mutants in IF2~ start site (that eliminate IF21~ expression).

lack of IF2AI, but rather reflected an inhibitory effect caused by the very high level of IF2AI protein synthesized from the unrepressed promotors, which was at least two-fold. It thus seems that an excess of wildtype IF2 as well as IF2A1, although at different concentrations, is inhibitory to growth. Since IF2 and EF-Tu binding sites on the ribosome overlap [28], an excess of IF2 (or IF2A1) could reduce the growth rate by preventing the binding of EF-Tu. Our results show that the large N-temfinal part of IF2 is not essential, but that the truncated form IF2AI seems to be less efficient in supporting growth and is thus required in higher concentrations. Moreover. the C-terminal part of IF2 is clearly essential in vivo. The major catalytic sites are thus present in the middle and C-terminal parts of the IF2 protein. The 55 000 Mr protein has the G-domain at its N-terminus which makes it more similar to other prokaryotic GTPbinding proteins (EF-Tu, EF-G, LepA, Era) and the eukaryotic Ras proteins. In vitro biological properties of IF2 fragments

It is obvious that an IF2 derived protein of molecular weight 55 000 or higher, as long as it contains the Cterminal half, will support growth of E coii and fulfill all the known functions of IF2. Indeed, a proteolysed form of IF2, IF2y (molecular weight 65 000) was equally or only slightly less active than IF2~ when tested in three in vitro assays characteristic for IF2 activity: fMet-tRNAfMet binding to 70S ribosomes, Nterminal dipeptide synthesis in a DNA-dependent transcription/translation system and ribosome dependent GTP hydrolysis [ 16]. In order to test the relevant biological activities of short fragments of IF2, we used'a gene fusion technique to synthesize stable fragments. The following strategy was applied [29]: 1) Subcloning of a defined IF2 gene segment in an expression vector in-frame with an ATG on its 5' side and lacZ in its 3' side. The fused gene was under the control of an inducible promotor. 2) Production of a fusion protein that could be rapidly purified by affinity chromatography. 3) Removal of the IF2 domain of the chimeric 13galactosidase hybrid protein by limited proteolysis with chymotrypsin. The low molecular weight fragment of IF2 is easily separated from the remainder of the fusion protein. A protein of an apparent i4 kDa mass was purified and its N- and C-termini were identified. The fragment has a calculated mass of 19.4 kDa and spans all the consensus sequences found in G domains [29]. This segment of IF2 can form binary complexes with GDP by Millipore filter assay and can be photo-

The structural and functionaldomains of E colt IF2 affinity cross-linked to GTP, therefore indicating directly for the first time that the fragment corresponds to the GTP binding domain. In GDP binding experiments, IF2 shows a maximum binding around 6-7 laM GDP; a 3-4 fold excess in GDP concentration is needed for the fragment to reach a GDP binding level approaching that obtained with intact IF2 [29]. The IF2 segment, while binding guanine nucleotide, does not exhibit GTPase activity in the presence or absence of ribosomes, as was found with the 65 kDa fragment of IF2. It is assumed that active centers of IF2 located outside the G domain are needed for the latter reaction. The DNA sequence of three other bacterial species of IF2 in addition to that of E colt are available, B stearothermophilus, B subtilis and S faecium. It is remarkable that the middle and C-terminal parts of the protein are highly conserved between the four proteins, whereas there is much divergence in the Nterminal region in accordance with the catalytic site being located in the C-terminal half of the molecule. Since the catalytic sites are in the C-terminal half of the molecule and since this C-terminal half (55 kDa protein) in physiological amounts is unable to support growth, the role of the N-terminal moiety of IF2 may be questioned. As an approach to elucidating the function of the N-terminal part of IF2 and more specifically the reason why two forms of the factor coexist in the cell, we constructed mutations that allow the expression of just one form of IF2 to the exclusion of the other [20].

1563

AUG ---) AUC (Met 165 ~ Ile). Both double mutants allow expression of IF2~x to the exclusion of IF2131/[~2 (fig 6).

Complementation of a null mutation In order to elucidate the significance of the existence of the two forms of IF2, we tested the constructions which synthesize only one of them for their ability to replace the chromosomal infB gene. A multicopy plasmid carrying a nusA-infB insert but lacking the main promoters of the metY-nusA-infB operon was used to synthesize IF2o~ and IF213 in physiological amounts. The deletion that eliminates IF2o~ expression and both double mutants of the IF21~1/152 start site that eliminate IF213 expression, were rec!oned in this plasmid (fig 6). The resulting plasmids were used to transform the strain previously descri0ed that contains a thermosensitive lysogenic )~infB and a chromosomal infB null mutation. After heat curing of )dnfB, surviving bacteria were obtained. The cured strains were shown to be stable through several purifications at 42°C. The presence of just one form of IF2 in the cured strains containing the corresponding infB mutant plasmid was confirmed by Western blot analysis (fig 5). Therefore, each one of the two forms of IF2 in near physiological amounts is sufficient for growth of E colt at 42°C. However, when tested at lower temperatures, the strains containing single forms of IF2 are distinctly retarded compared to parental strain. The retarded phenotype is visible at 37°C and is enhanced at 30°C, where the 'single form' strains are cold sensitive.

In vivo study of the two forms of initiation factor

IF2 (~ and ~)

Elimination of lF2ot synthesis A large deletion of the IF20~ specific part of infB gene was constructed between two restriction sites created by site-directed mutagenesis. This deletion eliminates both the IF2a ribosome binding site and most of the IF2a specific N-terminal coding region, but leaves the translational regulatory elements of the IF2131/[~2 start site (fig 6). A plasmid carrying this deletion expresses only the [~ (~1/~2) form of IF2.

Elimination of lF2 O synthesis In order to abolish IF213 expression, both initiation codons of IF2131 and IF21~2 were changed by sitedirected mutagenesis. In combination with the GUG ---> GUC mutation of IF2~l start codon, we constructed two mutations of the AUG start codon of IF2132 (Met 165 of IF2a): the first changed AUG ACG (Met 165 ~ Thr) and the second changed

Discussion and conclusion The construction of a strain (SL598R) carrying a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a )~ lysogenic phage allowed us to demonstrate that the infB gene is essential. The infB gone encodes two of the largest proteins involved in translation: the two forms of translation initiation factor IF2. The shorter form of IF2 (IF213) is identical to the 4/5 C-terminal part of the larger one (IF2a). In addition a third form of IF2, IF2y, can be generated by partial proteolysis during isolation of the factor. The in vitro study of this proteolytic fragment showed that the main catalytic centers of IF2 are located in the C-terminal 2/3 of the protein [16]. In vivo a shorter portion (55 kDa from the C-terminal) of IF2 was able to support growtb of E colt provided that it was supplied in excess [27]. The latter polypeptide (55 kDa) contains the G-domain and the C-terminal end of IF2. Separate studies of shorter fragments of

1564

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infB"

ISB" pBR322 " f /

Fig 6. Structure of a plasmid carrying a nusA-infB insert in pBR322 and 'single form of IF2" its derivatives. *above X indicate Xhoi restriction sites constructed by site-directed mutagenesis. Deletion A45 (that eliminates IF2ct synthesis) was obtained by deletion of the DNA between these two Xhol sites. *above a nucleotide shows the replacement obtained by site-directed mutagenesis in the different start codons of IF2l]l/~2. These mutations of IF21~l/132start sites resulted in elimination of IF21~1/[~2synthesis. the factor are under investigation and should provide a more detailed understanding of the main functions of IF2 in initiation of translation: stimulation of the binding of flVlet-tRNAr~et to the 30S ribosomal subunit and hydrolysis of GTP during formation of the 70S initiation complex. The location of the catalytic centers of IF2 in the Cterminal half of the protein brings up the question of the role of the N-terminal part and the functional significance of the two forms of IF2. Indeed, this Nterminal region of the factor is the one which makes the difference between IF2t~ and IF2[I. The 13 form arises from an internal translation initiation site in the infB gene. Two nearby functional start codons exist for IF2I~ and produce two related species IF2131 and

IF2~2 of the shorter form of IF2. The mechanism by which IF2[~1/~2 are expressed is mostly interesting: neither of the two start codons is preceded by a good SD sequence and the IF2131/~2 initiation sites are located inside a translated region of the infB mRNA. We observed that the IF2~ specific coding part of the gene contained more codons read by rare isoacceptor tRNA species [22]. Ribosomes pausing at these codons may induce some uncoupling of transcription to translation and therefore expose the IF2131/~2 start site. Alternatively, it may slow down translation of the IF2ct specific region in a way that sometimes prevents initiation of IF20~ and consequently makes the IF2~l/ 132 start site accessible. Nevertheless, IF2~1/[~2 are well expressed, and the shorter form of IF2 exists in the cell in a ratio 1/2 of IF2tx. We have shown that the two forms of IF2 are sufficient to support growth of E coli at 42°C when present in near physiological amounts, but that both forms are required for maximal growth, especially at lower temperatures. Other genes possessing an internal in-phase translation initiation site similar to infB have been described in the literature. This genetic organization exists in several phages whereas it is quite rare in bacteria. Three examples have been reported in E coil for the genes cheA [301, mcrB [31] and clpB [32]. Interestingly, CIpB is a heat-shock protein and a strain with a null mutation in clpB displays a slower growth rate at 44°C [32], whereas IF2 (both forms) is induced by cold shock [33] and our results show that the absence of one form causes cold sensitivity to the cell. An overlapping arrangement of genes is thought to be a general strategy in phage systems for efficient use of a limited coding capacity. This argument does not hold for bacteria and is also not sufficient to account for some phage organisation of genes [34]. An interesting example of two in-phase overlapping genes is given by the right repeat of transposon Tn5 [35, 36]. Opposite functions in transposition have been reported for the two proteins encoded by the right repeat: protein 1 is absolutely required for transposition, whereas protein 2 is competent to inhibit transposition. Isberg et al [35] have proposed two mechanisms for transposition regulation: protein 2, which is identical to protein 1 C-terminus could bind to some target sequences recognized by their common C-terminal domain and thus prevent protein 1 from binding. However, it would be unable to promote transposition which would necessitate the presence of the N-terminal specific domain of protein 1. Alternatively, proteins 1 and 2 could form oligomeric complexes, that unlike pure protein 1 oligomers, would not be active in transposition. In the case of IF2, the existence of differentiated roles for IF2t~ and IF2~ constitutes an unanswered question. Ishihama

The structural and functional domains of E co!i IF2 Table I. Growth on plates of the 'one-form' strains. 42°C (1)

37°C (1)

30°C (2)

Wild type E coli strain

++++

++++

++++

pBR322 with nusA-infB insert

++++

++++

+++

'IF213 only' strain

+++

+

_

'IF2tx only' strain a

+++

+

_

'IF2cz only' strain b

+++

+

_

3

4

++++ very good growth; +++ good growth but slightly smaller colonies; + microcolonies; - no isolated colonies; (1) after 16 h (at 37°C and 42°C); (2) after 20 h (at 30°C); astrain where start codon of IF2~I/1~2 has been changed: GTG --~ GTCand ATG --->ACG; bstrain where start codon of IF213111~2has been changed: GTG --->GTC and ATG ---> ATC.

5 6 7

8 [37] has reported that IF21~ (but not IF2~) was able to associate with R N A polymerase. He proposed a regulatory role for IF21~, as well as fMet-tRNAf~ t and EF-Tu in the coupling o f transcription to translation. By analogy with other genes containing an internal in-phase initiation site [34, 35], the existence o f oligomeric complexes of IF2o~ plus IF2I~ can be suggested. Such a hypothesis could be favored by recent data which indicate that the C-terminal domain o f IF2 harbors DNA-binding properties in vitro (Vachon e t al, to be published). Indeed, most DNA-binding proteins act as dimers or other multimers. In conclusion, the functional importance of the two forms o f IF2 is supported by the observation that they have been detected in other Enterobacteriaceae [38] and in the Gram-positive B a c i l l u s subtilis [8]. Some inessential function may have been conserved through evolution.

9 10 11

12 13 14 15

Acknowledgments We are grateful to N Gendron for aid with the manuscript. Thi.~ work was supported by grants from the following: CNRS (URA 1139), Contrat CEE No SCl*]0194-C(AM), Contrat de Recherche Externe INSERM No 891017, Fondation pour la Recherche Mrdicale to M Grunberg-Managa and S Laalami, Universit6 Paris 7 (Chapitre 66-71, Soutien de Programme).

16

17

References 1 Hershey JWB (1987) Protein Biosynthesis (Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE, eds), ASM, Washington, DC, 613-647 2 Shiba K, Ito K, Nakamura Y, Dondon J, GrunbergManago M (1986) Altered translational initiation factor 2

18

I_,o_,~=

in the cold sensitive ssvG mutant affects protein export in Escherichia coli. EMB'O J 5, 3001-3006 Butler JS, Springer M, Dondon J, Graffe M, GrunbergManago M (1986) Escherichia coli protein synthesis initiation factor IF3 controls its own gene expression at the translational level /n vivo. J Mol Biol 192, 767780 Plumbridge JA, Howe JG, Springer M, TouatiSchwartz D, Hershey JWB, Grunberg-Manago M (1982) Cloning and mapping of a gene for translational initiation factor IF2 in Escherichia coil Proc Natl Acad Sci USA 79, 5033-5037 Ishii S, Kuroki K, lmamoto F (1984) tRNAp_met gene in the leader region of the nusA operon in Escherichia coli. Proc Natl Acad Sci USA 81,409-413 Kurihara T, Nakamura Y (1983) Cloning of the nusA gene of Escherichia coli. Mol Gen Genet 190, 189-195 Sands JF, Regnier P, Cummings HS, GrunbergManago M, Hershey JWB (1988) The existence of two genes between infB and rpsO in the Escherichia coil genome: DNA sequencing and S1 nuclease mapping. Nucleic Acids. Res 16, 10803-10816 Shazand K, Tucker J, Chiang R, Stansmore K, SperlingPetersen HU, Grunberg-Manago M, Rabinowitz JC, Leighton T (1990) Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor IF2. J Bacteriol 172, 2675-2687 Bourne HR, Sanders DA, McCormick F (1990) The GTPase superfamily: a conserved switch for diverse cell functions. Nature (Lond) 348, 125-132 Jurnak F (1985) Structure of the GDP domain of EF-Tu and location of the aminoacids homologous to ras oncogene proteins. Science 230, 32-36 La Cour TFM, Nyborg J, Thirup S, Clark BFC (1985) Structural studies of the binding of elongation factor Tu from E coli as studied by X-ray crystallography. EMBO J 4, 2385-2388 Leberman R, Egner U (1984) Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21. EMBO J 3, 33%341 March PE, Inoute M (1985) Characterisation of the lep operon of Escherichia coil J Biol Chem 260, 7206--7213 Ahnn J, March PE, Takiff HE, Inouye M (1986) A GTPbinding protein of Escherichia coli has homology to yeast RAS proteins. Proe Natl Acad Sci USA 83, 8849-8853 Parmeggiani A, Swart GWM, Mortensen KK, Jensen M, Clark BFC, Dente L, Cortese R (1987) Properties of a genetically engineered G domain of elongation factor Tu. Proc Natl Acad Sci USA 84, 3141-3145 Cenatiempo Y, Deville F, Dondon J, Grunberg-Manago M, Sacerdot C, Hershey JWB, Hansen HE Petersen HU, Clark BFC, Kjeldgaard M, La Cour TFM, Mortensen KK, Nyborg J (1987) The protein synthesis initiation factor 2 G-domain. Study of a functionally active C-terminal 65kDa fragment of IF2 from Escherichia coli. Biochemisto' 26, 5070-5076 Dondon J, Plumbridge JA, Hershey JWB, GrunbergManago M (1985) Overproduction and purification of Initiation Factor IF2 and pNusA proteins from a recombinant plasmid bearing strain. Biochimie 67,643--649 Plumbridge JA, Deville F, Sacerdot C, Petersen P. Cenatiempo Y, Cozzone A, Grunberg-Manago M, Hershey JWB (1985) Two translational initiation sites in the infB gene are used to express initiation factor IF2et and IF2~ in Escherichia coli. EMBO J 4, 223-229

1566 Iq

20

21

22

23 24 25 26

27

S Laalami et at

MoreI-Deville F, Vachon G, Sacerdot C, Cozzone A, Grunbe~z-Manago M. Cenatiempo Y (1990) Characterisation of the translational start site for IF213, a short form of Escherichia coli initiation factor IF2. Eur J Biochem 188,605-614 Sacerdot C, Vachon G, Laalami S, Morel-Deville F, Cenatiempo Y, Grunberg-Manago M (1991) Both forms of translational Initiation Factor IF2 (~x and 13) are required for maximal growth of E coil Evidence for two translation initiation codons for IF2~. J Mol Biol (submitted) Mortensen KK, Nyengaard NR, Hershey JWB, Laalami S, Sperling-Petersen HU (1991) Superexpression and fast purification of E coli initiation factor IF2. Biochimie 73, 983-989 Sacerdot C, Dessen P, Hershey JWB, Plumbridge JA, Gnmbe~-Manago M (1984) Sequence of the initiation factor IF2 gene: Unusual protein features and homologies with elongation factors. Proc Nat! Acad Sci USA 81, 7787-7791 Sperling-Petersen HU, Mortensen KKA (1990) Structural model for initiation factor IF2 from E coli. Protein Eng 3, 343-344 Petersen HU (1985) Function of tRNA in initiation of procaryotic translation. Mat Fys Medd Dan Vid Selsk 41, 291-335 Lassen S, Mortensen KK, Sperling-Petersen HU (1991) J Bacteriol (submitted) McCormick F, Clark BFC, La Cour TFM, Kjelgaard M, Norskov-Lauristen L, Nyberg J (1985) A model for the tertiary structure of p21, the product of the ras oncogene. Science 230, 78-82 Laalami S, Putzer H, Plumbridge JA, Grunberg-Manago M (i991) A severly truncated form of translational Initiation Factor 2 supports growth of Escherichia coli. J Mol Biol 220, 335-349

28 29

30 31

32 33 34 35 36 37 38

Grunberg-Manago M, Dondon J, Graffe M (1972) Inhibition by thiostrepton of the IF2 dependent ribosomal GTPase. FEBS Lett 22, 217-221 Vachon G, Laalami S, Grunberg-Manago M, Julien R, Cenatiempo Y (1990) Purified internal G-domain of translational initiation factor 1F2 displays guanine nucleotide binding properties. Biochemistry 29, 9728- 9733 Smith RA, Parkinson JS (1980) Overlapping genes at the cheA locus of Escherichia coil Proc Natl Acad Sci USA 77, 5370--5374 Ross TK, Achberger EC, Braymer DH (1989) Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translation initiation sites at the mcrB locus. J Bacteriol 171, 1974- 1981 Squires CL, Pedersen S, Ross BM, Squires C (1991) CIpB is the Escherichia coli heat-shock protein F84.1. J Bacteriol 173, 4254--4262 Jones PG, Van Bogelen RA, Neidhardt FC (1987) Induction of proteins in response to low temperature in Escheriehia coli. J Bacteriol 169, 2092-2095 Shaw JE, Murialdo H (1980) Morphogenic genes C and Nu3 overlap in bacteriophage &. Nature (Lond) 283, 30--35 Isberg RR, Lazaar AL, Syvanen M (1982) Regulation of Tn5 by the right-repeat proteins: control at the level of the transposition reaction? Cell 30, 883-892 Johnson RC, Y'm JCP, Reznikoff WS (1982) Control of Tn5 transposition in Escherichia coil is mediated by protein from the right repeat. Cell 30, 873--882 Ishihama A (1988) Promoter selectivity of prokaryotic RNA polymerases. Trends Genet 4, 282-286 Howe JG, Hershey JWB (1984) The rate of evolutionary divergence of initiation factors IF2 and IF3 in various bacterial species determined quantitatively by immunoblotting. Arch Microbiol 140, 187192