A 'random steady-state' model for the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase enzyme complexes.

The multienzyme complexes, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, involved in the central metabolism of Escherichia coli consist of multiple copies of three different enzymes, E1, E2 and E3, that cooperate to channel substrate intermediates between their active sites. The E2 components form the core of the complex, while a mixture of E1 and E3 components binds to the core. We present a random steady-state model to describe catalysis by such multienzyme complexes. At a fast time scale, the model describes the enzyme catalytic mechanisms of substrate channeling at a steady state, by polynomially approximating the analytic solution of a biochemical master equation. At a slower time scale, the structural organization of the different enzymes in the complex and their random binding/unbinding to the core is modeled using methods from equilibrium statistical mechanics. Biologically, the model describes the optimization of catalytic activity by substrate sharing over the entire enzyme complex. The resulting enzymatic models illustrate the random steady state (RSS) for modeling multienzyme complexes in metabolic pathways.

DNA sequence and structure: direct and indirect recognition in protein-DNA binding.

MOTIVATION: Direct recognition, or direct readout, of DNA bases by a DNA-binding protein involves amino acids that interact directly with features specific to each base. Experimental evidence also shows that in many cases the protein achieves partial sequence specificity by indirect recognition, i.e., by recognizing structural properties of the DNA. (1) Could threading a DNA sequence onto a crystal structure of bound DNA help explain the indirect recognition component of sequence specificity? (2) Might the resulting pure-structure computational motif manifest itself in familiar sequence-based computational motifs? RESULTS: The starting structure motif was a crystal structure of DNA bound to the integration host factor protein (IHF) of E. coli. IHF is known to exhibit both direct and indirect recognition of its binding sites. (1) Threading DNA sequences onto the crystal structure showed statistically significant partial separation of 60 IHF binding sites from random and intragenic sequences and was positively correlated with binding affinity. (2) The crystal structure was shown to be equivalent to a linear Markov network, and so, to a joint probability distribution over sequences, computable in linear time. It was transformed algorithmically into several common pure-sequence representations, including (a) small sets of short exact strings, (b) weight matrices, (c) consensus regular patterns, (d) multiple sequence alignments, and (e) phylogenetic trees. In all cases the pure-sequence motifs retained statistically significant partial separation of the IHF binding sites from random and intragenic sequences. Most exhibited positive correlation with binding affinity. The multiple alignment showed some conserved columns, and the phylogenetic tree partially mixed low-energy sequences with IHF binding sites but separated high-energy sequences. The conclusion is that deformation energy explains part of indirect recognition, which explains part of IHF sequence-specific binding.

Activation and repression of transcription initiation by a distant DNA structural transition.

Negative superhelical tension can drive local transitions to alternative DNA structures. Long regions of DNA may contain several sites that are susceptible to forming alternative structures. Their relative propensities to undergo transition are ordered according to the energies required for their formation. These energies have two components - the energy needed to drive the transition and the energy relieved by the partial relaxation of superhelicity that the transition provides. This coupling can cause a complex competition among the possible transitions, in which the formation of one energetically favourable alternative structure may inhibit the formation of another within the same domain. In principle, DNA structural competitions can affect the structural and energetic requirements for the initiation of transcription at distant promoter sites. We have tested this possibility by examining the effects of structural transitions on transcription initiation from promoter sites in the same superhelical domain. Specifically, we describe the effects of the presence of a Z-DNA-forming DNA sequence on the basal levels of expression of two supercoiling-sensitive promoters of Escherichia coli, ilvPG and gyrA. We demonstrate transcriptional repression of the ilvPG promoter and activation of the gyrA promoter. We present evidence that this regulation is effected by the superhelically induced B- to Z-DNA transition in a manner that is both orientation and distance independent. We discuss the mechanism of topological coupling between left-handed Z-DNA and the regulation of promoter activity. We also discuss the possibility that the coupling of DNA structural transitions and transcriptional activity might be used as a general regulatory mechanism for gene expression.

The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons.

Transcriptional activities of closely spaced divergent promoters are affected by the accumulation of local negative superhelicity in the region between transcribing RNA polymerase molecules (transcriptional coupling). The effect of this transcription-induced DNA supercoiling on these promoters depends on their intrinsic properties. As the global superhelical density of the chromosome is controlled by the energy charge of the cell, which is affected by environmental stresses and transitions from one growth state to another, the transcriptional coupling that occurs between divergently transcribed promoters is likely to serve a physiological purpose. Here, we suggest that transcriptional coupling between the divergent promoters of the ilvYC operon of Escherichia coli serves to co-ordinate the expression of this operon with other operons of the ilv regulon during metabolic adjustments associated with growth state transitions. As DNA supercoiling-dependent transcriptional coupling between the promoters of other divergently transcribed operons is investigated, additional global gene regulatory mechanisms and physiological roles are sure to emerge.

Improved statistical inference from DNA microarray data using analysis of variance and a Bayesian statistical framework. Analysis of global gene expression in Escherichia coli K12.

We describe statistical methods based on the t test that can be conveniently used on high density array data to test for statistically significant differences between treatments. These t tests employ either the observed variance among replicates within treatments or a Bayesian estimate of the variance among replicates within treatments based on a prior estimate obtained from a local estimate of the standard deviation. The Bayesian prior allows statistical inference to be made from microarray data even when experiments are only replicated at nominal levels. We apply these new statistical tests to a data set that examined differential gene expression patterns in IHF(+) and IHF(-) Escherichia coli cells (Arfin, S. M., Long, A. D., Ito, E. T., Tolleri, L., Riehle, M. M., Paegle, E. S., and Hatfield, G. W. (2000) J. Biol. Chem. 275, 29672-29684). These analyses identify a more biologically reasonable set of candidate genes than those identified using statistical tests not incorporating a Bayesian prior. We also show that statistical tests based on analysis of variance and a Bayesian prior identify genes that are up- or down-regulated following an experimental manipulation more reliably than approaches based only on a t test or fold change. All the described tests are implemented in a simple-to-use web interface called Cyber-T that is located on the University of California at Irvine genomics web site.

DNA supercoiling-dependent transcriptional coupling between the divergently transcribed promoters of the ilvYC operon of Escherichia coli is proportional to promoter strengths and transcript lengths.

The twin-domain model of Liu and Wang suggested that high levels of DNA supercoiling generated in the region between closely spaced divergently transcribed promoters could serve to couple the activities of these promoters transcriptionally. In this report, we use topoisomer sets of defined superhelical densities as DNA templates in a purified in vitro transcription system to demonstrate transcriptional coupling between the divergently transcribed ilvY and ilvC promoters of the ilvYC operon of Escherichia coli. Current evidence for this type of DNA supercoiling-dependent transcriptional coupling, based largely on the in vivo activities of promoters contained in engineered DNA constructs, suggests that the transcription complex must be physically hindered to generate DNA supercoils and to prevent their diffusion throughout the DNA duplex. However, the in vitro results presented here demonstrate that (i) transcriptional coupling is observed between the divergent promoters of the ilvYC operon in the absence of transcript anchoring; (ii) the magnitude of the negative DNA supercoiling generated in the divergent promoter region is proportional to the sum of the global and transcription-induced superhelicity; and (iii) the magnitude of this transcription-induced superhelicity is proportional to promoter strengths and transcript lengths.

Global gene expression profiling in Escherichia coli K12. The effects of integration host factor.

We have used nylon membranes spotted in duplicate with full-length polymerase chain reaction-generated products of each of the 4,290 predicted Escherichia coli K12 open reading frames (ORFs) to measure the gene expression profiles in otherwise isogenic integration host factor IHF(+) and IHF(-) strains. Our results demonstrate that random hexamer rather than 3' ORF-specific priming of cDNA probe synthesis is required for accurate measurement of gene expression levels in bacteria. This is explained by the fact that the currently available set of 4,290 unique 3' ORF-specific primers do not hybridize to each ORF with equal efficiency and by the fact that widely differing degradation rates (steady-state levels) are observed for the 25-base pair region of each message complementary to each ORF-specific primer. To evaluate the DNA microarray data reported here, we used a linear analysis of variance (ANOVA) model appropriate for our experimental design. These statistical methods allowed us to identify and appropriately correct for experimental variables that affect the reproducibility and accuracy of DNA microarray measurements and allowed us to determine the statistical significance of gene expression differences between our IHF(+) and IHF(-) strains. Our results demonstrate that small differences in gene expression levels can be accurately measured and that the significance of differential gene expression measurements cannot be assessed simply by the magnitude of the fold difference. Our statistical criteria, supported by excellent agreement between previously determined effects of IHF on gene expression and the results reported here, have allowed us to identify new genes regulated by IHF with a high degree of confidence.

Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli.

The twin-domain model [Liu, L. F. & Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024-7027] suggests that closely spaced, divergent, superhelically sensitive promoters can affect the transcriptional activity of one another by transcriptionally induced negative DNA supercoiling generated in the divergent promoter region. This gene arrangement is observed for many LysR-type-regulated operons in bacteria. We have examined the effects of divergent transcription in the prototypic LysR-type system, the ilvYC operon of Escherichia coli. Double-reporter constructs with the lacZ gene under transcriptional control of the ilvC promoter and the galK gene under control of the divergent ilvY promoter were used to demonstrate that a down-promoter mutation in the ilvY promoter severely decreases in vivo transcription from the ilvC promoter. However, a down-promoter mutation in the ilvC promoter only slightly affects transcription from the ilvY promoter. In vitro transcription assays with DNA topoisomers showed that transcription from the ilvC promoter increases over the entire range of physiological superhelical densities, whereas transcription initiation from the ilvY promoter exhibits a broad optimum at a midphysiological superhelical density. Evidence that this promoter coupling is DNA supercoiling-dependent is provided by the observation that a novobiocin-induced decrease in global negative superhelicity results in an increase in ilvY promoter activity and a decrease in ilvC promoter activity predicted by the in vitro data. We suggest that this transcriptional coupling is important for coordinating basal level expression of the ilvYC operon with the nutritional and environmental conditions of cell growth.

Inhibition of DNA supercoiling-dependent transcriptional activation by a distant B-DNA to Z-DNA transition.

Negative DNA superhelicity can destabilize the local B-form DNA structure and can drive transitions to other conformations at susceptible sites. In a molecule containing multiple susceptible sites, superhelicity can couple these alternatives together, causing them to compete. In principle, these superhelically driven local structural transitions can be either facilitated or inhibited by proteins that bind at or near potential transition sites. If a DNA region that is susceptible to forming a superhelically induced alternate structure is stabilized in the B-form by a DNA-binding protein, its propensity for transition will be transferred to other sites within the same domain. If one of these secondary sites is in a promoter region, this transfer could facilitate open complex formation and thereby activate gene expression. We previously proposed that a supercoiling-dependent, DNA structural transmission mechanism of this type is responsible for the integration host factor-mediated activation of transcription from the ilvPG promoter of Escherichia coli (Sheridan, S. D., Benham, C. J. & Hatfield, G. W. (1998) J. Biol. Chem. 273, 21298-21308). In this report we confirm the validity of this mechanism by demonstrating the ability of a distant Z-DNA-forming site to compete with the superhelical destabilization that is required for integration host factor-mediated transcriptional activation, and thereby delay its occurrence.

Activation of gene expression by a novel DNA structural transmission mechanism that requires supercoiling-induced DNA duplex destabilization in an upstream activating sequence.

We have previously demonstrated that integration host factor (IHF)-mediated activation of transcription from the ilvPG promoter of Escherichia coli requires a supercoiled DNA template and occurs in the absence of specific interactions between IHF and RNA polymerase. In this report, we describe a novel, supercoiling-dependent, DNA structural transmission mechanism for this activation. We provide theoretical evidence for a supercoiling-induced DNA duplex destabilized (SIDD) structure in the A + T-rich, ilvPG regulatory region between base pair positions +1 and -160. We show that the region of this SIDD sequence immediately upstream of an IHF binding site centered at base pair position -92 is, in fact, destabilized by superhelical stress and that this duplex destabilization is inhibited by IHF binding. Thus, in the presence of IHF, the negative superhelical twist normally absorbed by this DNA structure in the promoter distal half of the SIDD sequence is transferred to the downstream portion of the SIDD sequence containing the ilvPG promoter site. This IHF-mediated translocation of superhelical energy facilitates duplex destabilization in the -10 region of the downstream ilvPG promoter and activates transcription by increasing the rate of open complex formation.

Activation of gene expression by a ligand-induced conformational change of a protein-DNA complex.

IlvY protein binds cooperatively to tandem operator sites in the divergent, overlapping, promoter-regulatory region of the ilvYC operon of Escherichia coli. IlvY positively regulates the expression of the ilvC gene in an inducer-dependent manner and negatively regulates the transcription of its own divergently transcribed structural gene in an inducer-independent manner. Although binding of IlvY protein to the tandem operators is sufficient to repress ilvY promoter-specific transcription, it is not sufficient to activate transcription from the ilvC promoter. Activation of ilvC promoter-specific transcription requires the additional binding of a small molecule inducer to the IlvY protein-DNA complex. The binding of inducer to IlvY protein does not affect the affinity of IlvY protein for the tandem operator sites. It does, however, cause a conformational change of the IlvY protein-DNA complex, which is correlated with the partial relief of an IlvY protein-induced bend of the DNA helix in the ilvC promoter region. This structural change in the IlvY protein-DNA complex results in a 100-fold increase in the affinity of RNA polymerase binding at the ilvC promoter site. The ability of a protein to regulate gene expression by ligand-responsive modulation of a protein-DNA structure is an emerging theme in gene regulation.

Growth rate-related regulation of the ilvGMEDA operon of Escherichia coli K-12 is a consequence of the polar frameshift mutation in the ilvG gene of this strain.

In Escherichia coli K-12 the intracellular levels of threonine deaminase and transaminase B, products of ilvA and ilvE, respectively, in the ilvGMEDA operon, increase with increasing growth rates (S. Pedersen, P. L. Bloch, S. Reeh, and F. C. Neidhardt, Cell 14:179-190, 1978). However, the transcriptional activities of the upstream ilvpG and the internal ilvpE promoters do not increase. Therefore, the growth rate-related expression of this operon is not regulated at the level of transcription initiation. Unlike other wild-type E. coli strains, E. coli K-12 contains a polar frameshift mutation in the ilvG gene (R. P. Lawther, D. H. Calhoun, C. W. Adams, C. A. Hauser, J. Gray, and G. W. Hatfield, Proc. Natl. Acad. Sci. USA 78:922-925, 1981). In an E. coli K-12 (IlvG+) derivative strain, where the reading frame of the ilvG gene is restored, no growth rate-related expression of the ilvGMEDA operon is observed. Thus, the growth rate-related expression of the ilvGMEDA operon in E. coli K-12 is the fortuitous consequence of the polar frameshift mutation in the ilvG gene of this strain.

Leucine-responsive regulatory protein-DNA interactions in the leader region of the ilvGMEDA operon of Escherichia coli.

The leucine-responsive regulatory protein (Lrp) regulates the expression of many operons in Escherichia coli including several involved in the metabolism of the branched-chain amino acids, L-isoleucine, L-valine, and L-leucine. The ilvGMEDA operon contains the genes for four of the five enzymes of the common pathway for the biosynthesis of these amino acids. A high affinity, consensus-like Lrp-DNA binding site has been identified at an unusual position in the leader region of this operon 226 base pairs downstream of the transcriptional initiation site between the attenuator and the ilvG gene. Binding to this site facilitates the cooperative binding of a second Lrp protomer to an adjacent, upstream, secondary site. At higher Lrp concentrations, binding to a third site is observed. Chemical, enzymatic, and alkylation protection and interference footprinting experiments demonstrate that the Lrp homodimer contacts the DNA helix at symmetrical half-sites present in adjacent major grooves and that the primary and secondary binding sites are separated by one helical turn and aligned along the same face of the DNA helix. In vivo, Lrp represses transcription through the leader-attenuator region of the ilvGMEDA operon. Lrp-dependent production of attenuated RNA transcripts is also observed in vitro. No transcriptional effects are observed, in vivo or in vitro, in the absence of an intact Lrp primary binding site. A possible physiological role for Lrp in the regulation of ilvGMEDA operon expression is discussed.

Effects of integration host factor and DNA supercoiling on transcription from the ilvPG promoter of Escherichia coli.

Integration host factor (IHF) activates transcription from the ilvPG promoter by severely distorting the DNA helix in an upstream region of a supercoiled DNA template in a way that alters the structure of the DNA in the downstream promoter region and facilitates open complex formation. In this report, the in vivo and in vitro influence of DNA supercoiling on transcription from this promoter is examined. In the absence of IHF, promoter activity increases with increased DNA supercoiling. In the presence of IHF, the same increases in superhelical DNA densities result in larger increases in promoter activity until a maximal activation of 5-fold is obtained. However, the relative transcriptional activities of the promoter in the presence and absence of IHF at any given DNA superhelical density remains the same. Thus, IHF and increased DNA supercoiling activate transcription by different mechanisms. Also, IHF binds with equal affinities to its target site on linear and supercoiled DNA templates. Therefore, IHF binding does not activate transcription simply by increasing the local negative supercoiling of the DNA helix in the downstream promoter region or by differential binding to relaxed and supercoiled DNA templates.

Transcriptional activation by protein-induced DNA bending: evidence for a DNA structural transmission model.

Integration host factor (IHF) is a DNA-bending protein that binds to an upstream activating sequence (UAS1) and, on a negatively supercoiled DNA template, activates transcription from the ilvPG promoter of the ilvG-MEDA operon of Escherichia coli. The transcriptional initiation site of the ilvGMEDA operon is located 92 bp downstream of UAS1. Activation is still observed when the orientation of the upstream IHF binding site is reversed. This manipulation places the IHF binding site on the opposite face of the DNA helix, directs the IHF-induced DNA bend in the opposite direction, and presents the opposite face of the nonsymmetrical, heterodimeric, IHF molecule to the downstream RNA polymerase. Lymphoid enhancer-binding factor, LEF-1, is a DNA-bending, lymphoid-specific, mammalian transcription factor that shares no amino acid sequence similarity with IHF. When the IHF site in UAS1 is replaced with a LEF-1 site, LEF-1 activates transcription from the downstream ilvPG promoter in E. coli as well as it is activated by its natural activator, IHF. These results suggest that specific interactions between IHF and RNA polymerase are not required for activation. The results of DNA structural studies show that IHF forms a protein-DNA complex in the UAS1 region that, in the absence of RNA polymerase, alters the structure of the DNA helix in the -10 hexanucleotide region of the downstream ilvPG promoter. The results of in vitro abortive transcription assays show that IIIF also increases the apparent rate of RNA polymerase isomerization from a closed to an open complex. We suggest, therefore, that IHF activates transcription by forming a higher-order protein-DNA complex in the UAS1 region that structurally alters the DNA helix in a way that facilitates open complex formation at the downstream ilvPG promoter site.

Codon pair utilization biases influence translational elongation step times.

Two independent assays capable of measuring the relative in vivo translational step times across a selected codon pair in a growing polypeptide in the bacterium Escherichia coli have been employed to demonstrate that codon pairs observed in protein coding sequences more frequently than predicted (over-represented codon pairs) are translated slower than pairs observed less frequently than expected (under-represented codon pairs). These results are consistent with the findings that translational step times are influenced by codon context and that these context effects are related to the compatabilities of adjacent tRNA isoacceptor molecules on the surface of a translating ribosome. These results also support our previous suggestion that the frequency of one codon next to another has co-evolved with the structure and abundance of tRNA isoacceptors in order to control the rates of translational step times without imposing additional constraints on amino acid sequences or protein structures.

DNA topology-mediated regulation of transcription initiation from the tandem promoters of the ilvGMEDA operon of Escherichia coli.

It is becoming increasingly clear that the intrinsic and protein-induced topological properties of the DNA helix influence transcriptional efficiency. In this report we describe the properties of two upstream activating regions that influence transcription from the non-overlapping tandem promoters of the ilvGMEDA operon of Escherichia coli. One 20 base-pair region between the promoter sites contains an intrinsic DNA bend that activates transcription from the downstream promoter. The other region contains an integration host factor (IHF) binding site that overlaps the upstream promoter site. IHF binding at this site represses transcription from the upstream promoter and enhances transcription from the downstream promoter. IHF also induces a severe bend in the DNA at its target binding site in the upstream promoter region. The activating property of the 20 base-pair DNA sequence located between the promoters is dependent upon the helical phasing of the sequence-directed DNA bend that it encodes. However, the IHF-mediated activation of transcription is not dependent upon the helical phasing (spatial orientation) of the upstream IHF and downstream promoter sites. The IHF-mediated activation of transcription is also uninfluenced by the presence or absence of the intrinsic DNA bend between its binding site and the downstream promoter site. These results suggest the interesting possibility that IHF activates transcription from the nearby downstream promoter simply by bending the DNA helix in the absence of specific IHF-RNA polymerase or upstream DNA-RNA polymerase interactions.

Integration host factor-mediated expression of the ilvGMEDA operon of Escherichia coli.

The structural genes of the ilvGMEDA operon of Escherichia coli are preceded by two promoters, ilvPG1 and ilvPG2, and a leader-attenuator region. Alkylation protection and hydroxyl radical footprinting techniques have been used to demonstrate that integration host factor (IHF) interacts with the nucleotides in a consensus-like DNA sequence located immediately downstream of the RNA polymerase transcriptional pause site in the leader-attenuator region. In the presence of purified IHF protein, in vitro transcriptional pausing of RNA polymerase at the leader-attenuator pause site is increased 2-fold and, concomitantly, a 2-fold increase in transcriptional termination at the attenuator is observed. Strains containing chromosomal transcriptional fusions of various segments of the ilvGMEDA promoter-attenuator region to the galK gene were used to show that IHF also decreases the in vivo basal level of transcriptional readthrough at the attenuator 2-fold. The binding of IHF to another target site in the ilvPG1 promoter region represses transcription from this promoter and causes a 4-fold stimulation of transcription initiation from the downstream ilvPG2 promoter 4-fold. This IHF-mediated control of transcription initiation from the upstream promoter region is independent of the regulation of transcription termination effected by IHF interaction at the attenuator site. Thus, IHF is capable of regulating the expression of the ilvGMEDA operon in opposing manners; it can activate transcription initiation of this operon from the ilvPG2 promoter 4-fold and increase the termination of this transcription at the downstream attenuator 2-fold.

Characterization of the integration host factor binding site in the ilvPG1 promoter region of the ilvGMEDA operon of Escherichia coli.

The ilvGMEDA operon of Escherichia coli, which encodes four of the five enzyme activities required for the biosynthesis of isoleucine and valine, is preceded by tandem promoters ilvPG1 and ilvPG2 which are separated by 72 base pairs. While both of these promoters are transcriptionally active in vitro, only the operon proximal promoter, ilvPG2, is transcriptionally active in vivo, and upstream DNA sequences encoding the ilvPG1 promoter region enhance the in vivo transcriptional activity of the ilvPG2 promoter 60-fold. The binding of the integration host factor protein (IHF) to this upstream region (Tsui, P., and Freundlich, M. (1989) J. Mol. Biol. 203, 817-820) has been shown to repress transcription from the ilvPG1 promoter both in vivo and in vitro (Pereira, R. F., Ortuno, M. J., and Lawther, R. P. (1988) Nucleic Acids Res. 16, 5972-5989). Furthermore, E. coli strains deficient for IHF are compromised for isoleucine and valine biosynthesis (Friden, P., Voelkel, K., Sternglantz, R., and Freundlich, M. (1984) J. Mol. Biol. 172, 573-579). Therefore, in order to further understand this repressor/activator role of IHF, we have undertaken a detailed analysis of the interaction of IHF with the DNA sequences in the ilvPG1 promoter region. The results of hydroxyl radical footprinting, dimethyl sulfate protection, and ethylation interference experiments show that IHF binds to a target site that overlaps the ilvPG1 promoter region. The results of these experiments also demonstrate that IHF interacts primarily with the minor groove of the DNA helix and that the IHF target site in the ilvPG1 promoter region shares a high degree of DNA sequence identity with other high affinity IHF target sites involved in DNA replication and site-specific recombination.

The primary structure of a halorhodopsin from Natronobacterium pharaonis. Structural, functional and evolutionary implications for bacterial rhodopsins and halorhodopsins.

We cloned and sequenced the gene coding for the polypeptide of a halorhodopsin in Natronobacterium pharaonis (named here pharaonis halorhodopsin). Peptide sequencing of cyanogen bromide fragments, and immunoreactions of the protein and synthetic peptides derived from the COOH-terminal gene sequence, confirmed that the open reading frame is the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences, as well as those for other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences (mutations/nucleotide at codon positions which do not result in amino acid changes) were calculated. These indicate very considerable evolutionary distance between each pair of genes. In spite of this, the three protein sequences show extensive similarities, indicating strong selective pressures. Conserved and conservatively replaced amino acid residues in all three proteins identify general features essential for ion-motive bacterial rhodopsins, responsible for overall structure and chromophore properties. Comparison of the bacteriorhodopsin sequence with those of the two halorhodopsins, on the other hand, identifies features involved in their specific (proton and chloride ion) transport functions.