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A simple method is presented for reconstructing phylogenetic trees on the basis of gene transposition. It is shown that differences in gene arrangements among genomes could allow us to determine whether a gene transposition event has occurred before or after species divergence from parsimonious considerations. The method is applied to evolutionary relationships among the bacterial class Proteobacteria, for which complete genomic sequences most densely accumulate and comprehensive gene order comparisons are possible. We were able to infer the emergence order of proteobacterial subclasses as epsilon-->beta-->gamma. This order is consistent with sequence-based inferences, which conversely confirms the usefulness of the approach presented here., (Copyright 2001 Academic Press.)

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.

Initiation of protein biosynthesis in bacteria requires three initiation factors: initiation factor 1, initiation factor 2 and initiation factor 3. The mechanism by which initiation factors form the 70S initiation complex with initiator fMet-tRNA(fMet) interacting with the initiation codon in the ribosomal P site and the second mRNA codon exposed in the A site is not yet understood. Here, we present a model for the function of initiation factors 1 and 2 that is based on the analysis of sequence homologies, biochemical evidence and the present knowledge of the three-dimensional structures of translation factors and ribosomes. The model predicts that initiation factors 1 and 2 interact with the ribosomal A site mimicking the structure of the elongation factor G. We present data that extend the mimicry hypothesis to initiation factors 1 and 2, originally postulated for the aminoacyl-tRNA x elongation factor Tu x GTP ternary complex, elongation factor G and release factors.

The binding of initiation factors to 30 S ribosomal subunits protects specific sets of nucleotides in 16 S rRNA from base-specific chemical probes. Initiation factor 3 (IF-3) protects residues G700, G703 and G791 from attack by kethoxal. These protected bases are close to those in 16 S rRNA that are protected by 50 S subunits, providing a structural basis for the subunit dissociation activity of IF-3. The IF-3-dependent protections also flank bases that are protected by P-site-bound tRNA, in keeping with the possibility that IF-3 may interact with initiator tRNA, or influence the properties of the 30 S P site during initiation. IF-1 protects G530, A1492 and A1493 and causes enhanced reactivity of A1408. These bases are precisely the ones that are protected by the binding of tRNA to the ribosomal A site. This suggests that IF-1 mimics A-site-bound tRNA, and could serve to prevent premature binding of aminoacyl tRNA by blocking the 30 S A site. We were unable to detect any effect of IF-2 on the reactivity pattern of 16 S rRNA, suggesting that this factor may interact primarily through protein-protein interactions.

Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood. Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA. Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2 alpha chains. The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology. The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function. The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2 alpha may bind to RNA.

An inhibitor of elongation factor G (EF-G) GTPase isolated from the ribosome wash of Escherichia coli was shown to stimulate the poly(A,U,G)- and initiation factor 2 (IF2)-dependent binding of N-formyl-[35S]Met-tRNAfMet to ribosomes. In the presence of saturating amounts of the EF-G GTPase inhibitor, neither addition of initiation factor 1 (IF1) nor addition of initiation factor 3 (IF3) caused a further stimulation of the formation of N-formyl-[35S]Met-tRNAfMET/poly(A,U,G)/ribosome complexes. Both IF1 and IF3 were shown to inhibit ribosome-dependent EF-G GTPase, especially when both initiation factors were added either in absence or in the presence of initiation factor 2 (IF2), poly(A,U,G) and N-formyl-Met-tRNAfMet. Therefore, we conclude that the EF-G GTPase inhibitor consisting of two polypeptide subunits with apparent molecular masses of 23,000 and 10,000 Da is a complex of initiation factors IF1 and IF3. The inhibition of EF-G GTPAse by IF3, but not the effects of IF1 in the presence or absence of IF3 could be reversed by increasing the Mg(2+)-concentration as already shown for the EF-G GTPase inhibitor. Therefore, IF1 as well as the EF-G GTPase inhibitor do not influence the ribosome-dependent EF-G GTPase by affecting the association of ribosomal subunits.

A method is presented for the detection in crude lysates of subnanogram amounts of proteins covalently bound to a specific nucleic acid sequence. The sensitivity of this method enabled us to study proteins cross-linked to specific DNA and mRNA sequences by irradiation of intact Escherichia coli cells with ultraviolet light. Among the proteins cross-linked to pBR322 DNA, the single strand binding protein, the HU-proteins, and the RNA polymerase beta and sigma subunits were present. Some, but not all proteins were cross-linked to 5-bromodeoxyuridine-substituted DNA more efficiently than to normal DNA. Ribosomal protein S1 is by far the most prominent protein cross-linked to mRNAs. Among the proteins cross-linked in smaller amounts to mRNAs are translation initiation factor IF 1, and at least six proteins of the 30 S ribosomal subunit, among which is S21. No 50 S proteins, nor IF-2, IF-3 or any of the elongation factors could be detected. Some UV-induced nucleic acid-protein cross-links were found to be heat-labile. It is concluded that the method employed may be used to compare the proteins interacting with different mRNAs, as well as single-copy DNA sequences from bacteria and eucaryotes with low complexity genomes.

Proteins cross-linked to pBR322 mRNAs and DNA by formaldehyde treatment of intact Escherichia coli cells have been detected with the use of a novel detection method. Among the proteins cross-linked to pBR322 mRNAs were S1, S21, and at least six other proteins of the small ribosomal subunit, initiation factor 1, elongation factor (EF) Tu, and very small amounts of EF-G and EF-Ts. The single strand binding protein, the HU-proteins, and RNA polymerase subunits alpha and beta were among the proteins cross-linked to pBR322 DNA. The results obtained suggest that the procedures described, can also be used to study interactions between different nucleic acid-bound polypeptides. The results are discussed in relation to the working mechanism of formaldehyde, and are compared to the results obtained with cross-linking induced by ultraviolet light. The methods presented should also be of use for the study of nucleic acid-protein interactions in other organisms.