Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection.

The objective of this study was to identify the mitochondrial proteins that undergo changes in phosphorylation during global ischemia and reperfusion in the isolated rabbit heart. We also assessed whether the cardioprotective intervention of ischemic preconditioning affected mitochondrial protein phosphorylation. We established a reconstituted system using isolated mitochondria and cytosol from control or ischemic hearts. We found that phosphorylation of a 46-kDa protein on a serine residue was increased in ischemia and that phosphorylation was reduced in control or preconditioned hearts. Using 2D gel electrophoresis and mass spectrometry, we have identified the 46-kDa protein as mitochondrial translational elongation factor Tu (EF-Tu(mt)). These data reveal that ischemia and preconditioning modulate the phosphorylation of EF-Tu(mt) and suggest that the mitochondrial protein synthesis machinery may be regulated by phosphorylation. Phosphorylation of mitochondrial EF-Tu has not been previously described; however, in prokaryotes, EF-Tu phosphorylation inhibits protein translation. We hypothesized that phosphorylation of mitochondrial EF-Tu would inhibit mitochondrial protein translation and attempted to reproduce the effect with inhibition of mitochondrial protein synthesis by chloramphenicol. We found that chloramphenicol pretreatment significantly reduced infarct size, suggesting that mitochondrial protein synthesis is one determinant of myocardial injury during ischemia and reperfusion.

The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present.

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.

Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approach.

Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.

The small subunit of the mammalian mitochondrial ribosome. Identification of the full complement of ribosomal proteins present.

Identification of all the protein components of the small subunit (28 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 28 S subunits followed by analysis of the resultant peptides by liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptide sequence information was used to search the human EST data bases and complete coding sequences of the proteins were assembled. The human mitochondrial ribosome has 29 distinct proteins in the small subunit. Fourteen of this group of proteins are homologs of the Escherichia coli 30 S ribosomal proteins S2, S5, S6, S7, S9, S10, S11, S12, S14, S15, S16, S17, S18, and S21. All of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. Surprisingly, three variants of ribosomal protein S18 are found in the mammalian and D. melanogaster mitochondrial ribosomes while C. elegans has two S18 homologs. The S18 homologs tend to be more closely related to chloroplast S18s than to prokaryotic S18s. No mitochondrial homologs to prokaryotic ribosomal proteins S1, S3, S4, S8, S13, S19, and S20 could be found in the peptides obtained from the whole 28 S subunit digests or by analysis of the available data bases. The remaining 15 proteins present in mammalian mitochondrial 28 S subunits (MRP-S22 through MRP-S36) are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of these proteins have a clear homolog in D. melanogaster while all but three can be found in the genome of C. elegans. Five of the mitochondrial specific ribosomal proteins have homologs in S. cerevisiae.

A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins.

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.

Interaction of mammalian mitochondrial elongation factor EF-Tu with guanine nucleotides.

Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts). Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tu(mt)) with guanine nucleotides was the focus of the work reported here. Equilibrium dialysis with [3H]GDP was used to measure the equilibrium dissociation constant of the EF-Tu(mt) x GDP complex (K(GDP) = 1.0 +/- 0.1 microM). Competition of GTP with a fluorescent derivative of GDP (mantGDP) for binding to EF-Tu(mt) was used to measure the dissociation constant of the EF-Tu(mt) x GTP complex (K(GTP) = 18 +/- 9 microM). The analysis of these data required information on the dissociation constant of the EF-Tu(mt) x mantGDP complex (K(mGDP) = 2.0 +/- 0.5 microM), which was measured by equilibrium dialysis. Both K(GDP) and K(GTP) for EF-Tu(mt) are quite different (about two orders of magnitude higher) than the dissociation constants of the corresponding complexes formed by Escherichia coli EF-Tu. The forward and reverse rate constants for the association and dissociation of the EF-Tu(mt) x GDP complex were determined using the change in the fluorescence of mantGDP upon interaction with EF-Tu(mt). These values are in agreement with a simple equilibrium binding interaction between EF-Tu(mt) and GDP. The results obtained are discussed in terms of the recently described crystal structure of the EF-Tu(mt) x GDP complex.

A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins.

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.

Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and elongation factor Ts.

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu.GTP.aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu.GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu.Ts). Mammalian mitochondrial EF-Tu (EF-Tu(mt)) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tu(mt) subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K(tRNA)) and for the dissociation of the EF-Tu.Ts(mt) complex (K(Ts)) were investigated. The equilibrium dissociation constant for the ternary complex was 18 +/- 4 nm, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 x 10(-)(4) s(-)(1), which is essentially equivalent to that observed for the ternary complex in Escherichia coli. The binding of EF-Tu(mt) to EF-Ts(mt) is mutually exclusive with the formation of the ternary complex. K(Ts) was determined by quantifying the effects of increasing concentrations of EF-Ts(mt) on the amount of ternary complex formed with EF-Tu(mt). The value obtained for K(Ts) (5.5 +/- 1.3 nm) is comparable to the value of K(tRNA).

Characterization and tRNA recognition of mammalian mitochondrial seryl-tRNA synthetase.

Animal mitochondrial protein synthesis systems contain two serine tRNAs (tRNAs(Ser)) corresponding to the codons AGY and UCN, each possessing an unusual secondary structure; the former lacks the entire D arm, and the latter has a slightly different cloverleaf structure. To elucidate whether these two tRNAs(Ser) can be recognized by the single animal mitochondrial seryl-tRNA synthetase (mt SerRS), we purified mt SerRS from bovine liver 2400-fold and showed that it can aminoacylate both of them. Specific interaction between mt SerRS and either of the tRNAs(Ser) was also observed in a gel retardation assay. cDNA cloning of bovine mt SerRS revealed that the deduced amino acid sequence of the enzyme contains 518 amino acid residues. The cDNAs of human and mouse mt SerRS were obtained by reverse transcription-polymerase chain reaction and expressed sequence tag data base searches. Elaborate inspection of primary sequences of mammalian mt SerRSs revealed diversity in the N-terminal domain responsible for tRNA recognition, indicating that the recognition mechanism of mammalian mt SerRS differs considerably from that of its prokaryotic counterpart. In addition, the human mt SerRS gene was found to be located on chromosome 19q13.1, to which the autosomal deafness locus DFNA4 is mapped.

High resolution crystal structure of bovine mitochondrial EF-Tu in complex with GDP.

The crystal structure of bovine mitochondrial elongation factor Tu (EF-Tu) in complex with GDP has been determined at a resolution of 1. 94 A. The structure is similar to that of EF-Tu:GDP from Escherichia coli and Thermus aquaticus, but the orientation of the GDP-binding domain 1 is changed relative to domains 2 and 3. Sixteen conserved water molecules common to EF-Tu and other G-proteins in the GDP-binding site are described. These water molecules create a network linking separated parts of the binding pocket. Mitochondrial EF-Tu binds nucleotides less tightly than prokaryotic EF-Tu possibly due to an increased mobility in regions close to the GDP-binding site. The C-terminal extension of mitochondrial EF-Tu has structural similarities with DNA recognising zinc fingers suggesting that the extension may be involved in recognition of RNA.

Expression and characterization of the human mitochondrial leucyl-tRNA synthetase.

A cDNA clone encoding the human mitochondrial leucyl-tRNA synthetase (mtLeuRS) has been identified from the EST databases. Analysis of the protein encoded by this cDNA indicates that the protein is 903 amino acids in length and contains a mitochondrial signal sequence that is predicted to encompass the first 21 amino acids. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl-tRNA synthetases and regions of high homology to other mitochondrial and bacterial LeuRS proteins. The mature form of this protein has been cloned and expressed in Escherichia coli. Gel filtration indicates that human mtLeuRS is active in a monomeric state, with an apparent molecular mass of 101 kDa. The human mtLeuRS is capable of aminoacylating E. coli tRNA(Leu). Its activity is inhibited at high levels of either monovalent or divalent cations. K(M) and k(cat) values for ATP:PP(i) exchange and for the aminoacylation reaction have been determined.

Identification of a mammalian mitochondrial homolog of ribosomal protein S7.

Bovine mitochondrial small subunit ribosomal proteins were separated by two-dimensional electrophoresis. The region containing the most basic protein(s) was excised and the protein(s) present subjected to in-gel digestion with trypsin. Electrospray tandem mass spectrometry was used to provide sequence information on some of the peptide products. Searches of the human EST database using the sequence of the longest peptide analyzed indicated that this peptide was from the mammalian mitochondrial homolog of prokaryotic ribosomal protein S7 (MRP S7(human)). MRP S7(human) is a 28-kDa protein with a pI of 10. Significant homology to bacterial S7 is observed especially in the C-terminal half of the protein. Surprisingly, MRP S7(human) shows less homology to the corresponding mitochondrial proteins from plants and fungi than to bacterial S7.

Effects of domain exchanges between Escherichia coli and mammalian mitochondrial EF-Tu on interactions with guanine nucleotides, aminoacyl-tRNA and ribosomes.

Escherichia coli elongation factor (EF-Tu) and the corresponding mammalian mitochondrial factor, EF-Tumt, show distinct differences in their affinities for guanine nucleotides and in their interactions with elongation factor Ts (EF-Ts) and mitochondrial tRNAs. To investigate the roles of the three domains of EF-Tu in these differences, six chimeric proteins were prepared in which the three domains were systematically switched. E. coli EF-Tu binds GDP much more tightly than EF-Tumt. This difference does not reside in domain I alone but is regulated by interactions with domains II and III. All the chimeric proteins formed ternary complexes with GTP and aminoacyl-tRNA although some had an increased or decreased activity in this assay. The activity of E. coli EF-Tu but not of EF-Tumt is stimulated by E. coli EF-Ts. The presence of any one of the domains of EF-Tumt in the prokaryotic factor reduced its interaction with E. coli EF-Ts 2-3-fold. In contrast, the presence of any of the three domains of E. coli EF-Tu in EF-Tumt allowed the mitochondrial factor to interact with bacterial EF-Ts. This observation indicates that even domain II which is not in contact with EF-Ts plays an important role in the nucleotide exchange reaction. EF-Tsmt interacts with all of the chimeras produced. However, with the exception of domain III exchanges, it inhibits the activities of the chimeras indicating that it could not be productively released to allow formation of the ternary complex. The unique ability of EF-Tumt to promote binding of mitochondrial Phe-tRNAPhe to the A-site of the ribosome resides in domains I and II. These studies indicate that the interactions of EF-Tu with its ligands is a complex process involving cross-talk between all three domains.

Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase.

Human mitochondrial phenylalanyl-tRNA synthetase (mtPheRS) has been identified from the human EST database. Using consensus sequences derived from conserved regions of the alpha and beta-subunits from bacterial PheRS, two partially sequenced cDNA clones were identified. Unexpectedly, sequence analysis indicated that one of these clones was a truncated form of the other. Detailed analysis indicates that unlike the (alphabeta)2 structure of the prokaryotic and eukaryotic cytoplasmic forms of PheRS, the human mtPheRS consists of a single polypeptide chain. This protein has been cloned and expressed in Escherichia coli. Gel filtration and analytical velocity sedimentation centrifugation indicate that the human mtPheRS is active in a monomeric form. The N-terminal 314 amino acid residues appear to be analogous to the alpha-subunit of the prokaryotic PheRS, while the C-terminal 100 amino acid residues correspond to a region of the beta-subunit known to interact with the anticodon of tRNAPhe. Comparisons with the sequences of PheRS from yeast and Drosophila mitochondria indicate they are 42 % and 51 % identical with the human mtPheRS, respectively. Sequence analysis confirms the presence of motifs characteristic of class II aminoacyl-tRNA synthetases. KM and kcat values for ATP:PPi exchange and for the aminoacylation reaction carried out by human mtPheRS have been determined. Evolutionary origins of this small monomeric human mtPheRS are unknown, however, implications are that this enzyme is a result of the simplification of the more complex (alphabeta)2 bacterial PheRS in which specific functional regions were retained.

Identification and cloning of human mitochondrial translational release factor 1 and the ribosome recycling factor.

The complete sequence of the cDNA for the coding region of human mitochondrial translational release factor 1 has been obtained from human EST clones and 5'RACE. This sequence has been analyzed to provide insights into the relationship between release factors from bacteria and from mammalian mitochondria. The complete cDNA for the human mitochondrial ribosome recycling factor has been assembled using EST clones. This factor has been expressed in Escherichia coli and purified as a His-tagged protein.

Roles of residues in mammalian mitochondrial elongation factor Ts in the interaction with mitochondrial and bacterial elongation factor Tu.

The crystal structure of the complex between Escherichia coli elongation factors Tu and Ts (EF-Tu.Ts) and subsequent mutagenesis work have provided insights into the roles of a number of residues in E. coli EF-Ts in its interaction with EF-Tu. The corresponding residues in bovine mitochondrial EF-Ts (EF-Tsmt) have been mutated. The abilities of the resulting EF-Tsmt derivatives to stimulate the activities of both E. coli and mitochondrial EF-Tu have been tested. Mutation of several residues in EF-Tsmt corresponding to amino acids important for the activity of E. coli EF-Ts has little or no effect on the activity of the mitochondrial factor, suggesting that these factors may use somewhat different mechanisms to promote guanine nucleotide exchange. In general, mutations that reduce the strength of the interaction between EF-Tsmt and E. coli EF-Tu increase the ability of EF-Tsmt to stimulate the activity of the bacterial factor. In contrast, these mutations tend to reduce the ability of EF-Tsmt to stimulate the activity of EF-Tumt. For example, F19A/I20A and H176A derivatives of EF-Tsmt are as active as E. coli EF-Ts in simulating E. coli EF-Tu. However, these mutations significantly decrease the ability of EF-Tsmt to stimulate EF-Tumt.

Mammalian mitochondrial methionyl-tRNA transformylase from bovine liver. Purification, characterization, and gene structure.

The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography. The polypeptide responsible for MTFmt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTFmt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTFmt consists of 357 amino acid residues. This sequence is about 30% identical to the corresponding Escherichia coli and yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTFmt formylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNAfMet) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNAmMet) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator Met-tRNAs. These observations are discussed in terms of the presence of a single tRNAMet gene in mammalian mitochondria.

Regulation of the activity of chloroplast translational initiation factor 3 by NH2- and COOH-terminal extensions.

The mature form of the chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis consists of an internal region homologous to prokaryotic IF3 flanked by long NH2- and COOH-terminal extensions. Sequences in these extensions reduce the activity of the homology domain in promoting initiation complex formation with chloroplast mRNAs and 30 S ribosomal subunits. A series of deletions of the NH2- and COOH-terminal extensions of IF3chl were constructed and tested for their effects on the activity of the homology domain. About half of the inhibitory effect arises from sequences within 9 residues of the junction between the NH2-terminal extension and the homology domain. The remaining inhibitory effect is the result of sequences in the COOH-terminal extension. The equilibrium constant governing the binding of the homology domain of IF3chl to 30 S subunits is estimated to be 1.3 x 10(7) M-1. Sequences close to the junction of the NH2-terminal extension and the homology domain reduce this binding constant about 10-fold. Sequences in the COOH-terminal extension have a similar negative effect. The negative effects of these two regions are cumulative, resulting in a 100-fold reduction of the binding constant. The 9 residues at the NH2-terminal extension effectively prevent the proofreading activity of IF3chl. The entire COOH-terminal extension reduces the proofreading ability by about half. These results are discussed in terms of the proposed three-dimensional structure of the homology domain of IF3chl.

Mutational analysis of the roles of residues in Escherichia coli elongation factor Ts in the interaction with elongation factor Tu.

The crystal structure of the Escherichia coli elongation factor (EF)-Tu.Ts complex indicates that there are extensive contacts between EF-Tu and EF-Ts. To determine the importance of these contacts in the interaction between E. coli EF-Tu and EF-Ts, residues in EF-Ts at the interface of these two proteins were mutated. The binding constants governing the interaction of the resulting EF-Ts variants with E. coli EF-Tu were determined. The effects of these mutations on the ability of EF-Ts to stimulate GDP exchange with EF-Tu.GDP and on its ability to stimulate the activity of EF-Tu in polymerization were tested. The results indicate that Arg-12, Met-19, and Met-20 in the N-terminal domain of EF-Ts and His-147 and Lys-166 and/or His-167 in subdomain C of EF-Ts are crucial in the interaction between EF-Tu and EF-Ts. Lys-23, Val-234, Met-235, and the C-terminal helix h13 are less important. The binding constants of the EF-Ts variants governing their interactions with EF-Tu correlate well with their activities in stimulating GDP exchange with EF-Tu. Mutations prepared in EF-Tu indicate that His-19 and Gln-114 but not Glu-348 in EF-Tu are moderately important for its interaction with EF-Ts.

Structural and mechanistic studies on chloroplast translational initiation factor 3 from Euglena gracilis.

Chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis contains a central region (homology domain) that is homologous to prokaryotic IF3. The homology domain is preceded by a long NH2-terminal extension (head), and followed by a 64 amino acid COOH-terminal extension (tail). Sequences in these extensions reduce the activity of the homology domain. To gain insight into these effects, a possible three-dimensional structure for the homology region of IF3chl has been modeled using the X-ray coordinates from the N- and C-domains of Bacillus stearothermophilus IF3. In B. stearothermophilus IF3, these two compact domains are thought to fold independently and are separated by a helical lysine-rich linker. The modeled structure suggests that IF3chl has a similar overall fold although some subtle differences are predicted to occur. Both the head and tail regions of IF3chl are oriented toward the linker region in the homology domain where they may potentially interfere with its function. Circular dichroism spectropolarimetry (CD) indicates that the lysine-rich linker region in IF3chl is not in a helical conformation and is probably a random coil. CD analysis indicates that a portion of the tail region of IF3chl is helical and that the tail has a direct interaction with the linker region in the homology domain. Site-directed mutagenesis of the linker indicates that two conserved lysine residues are important for the function of IF3chl and play a role in the binding of IF3chl to the 30S ribosomal subunit. Mutation of these residues affects the interaction of the homology domain with the tail.