The genetic code is degenerate. Each amino acid is encoded by up to six synonymous codons; the choice between these codons influences gene expression. Here, we show that in coding sequences, once a particular codon has been used, subsequent occurrences of the same amino acid do not use codons randomly, but favor codons that use the same tRNA. The effect is pronounced in rapidly induced genes, involves both frequent and rare codons and diminishes only slowly as a function of the distance between subsequent synonymous codons. Furthermore, we found that in S. cerevisiae codon correlation accelerates translation relative to the translation of synonymous yet anticorrelated sequences. The data suggest that tRNA diffusion away from the ribosome is slower than translation, and that some tRNA channeling takes place at the ribosome. They also establish that the dynamics of translation leave a significant signature at the level of the genome.

Symbiotic N 2 fixation in Bradyrhizobium japonicum is controlled by a complex transcription factor network. Part of it is a hierarchically arranged cascade in which the two-component regulatory system FixLJ, in response to a moderate decrease in oxygen concentration, activates the fixK 2 gene. The FixK 2 protein then activates not only a number of genes essential for microoxic respiration in symbiosis (fixNOQP and fixGHIS) but also further regulatory genes (rpoN 1 , nnrR, and fixK 1 ). The results of transcriptome analyses described here have led to a comprehensive and expanded definition of the FixJ, FixK 2 , and FixK 1 regulons, which, respectively, consist of 26, 204, and 29 genes specifically regulated in microoxically grown cells. Most of these genes are subject to positive control. Particular attention was addressed to the FixK 2 -dependent genes, which included a bioinformatics search for putative FixK 2 binding sites on DNA (FixK 2 boxes). Using an in vitro transcription assay with RNA polymerase holoenzyme and purified FixK 2 as the activator, we validated as direct targets eight new genes. Interestingly, the adjacent but divergently oriented fixK 1 and cycS genes shared the same FixK 2 box for the activation of transcription in both directions. This recognition site may also be a direct target for the FixK 1 protein, because activation of the cycS promoter required an intact fixK 1 gene and either microoxic or anoxic, denitrifying conditions. We present evidence that cycS codes for a c-type cytochrome which is important, but not essential, for nitrate respiration. Two other, unexpected results emerged from this study: (i) specifically FixK 1 seemed to exert a negative control on genes that are normally activated by the N 2 fixation-specific transcription factor NifA, and (ii) a larger number of genes are expressed in a FixK 2 -dependent manner in endosymbiotic bacteroids than in culture-grown cells, pointing to a possible symbiosisspecific control.

Hierarchically organized regulatory proteins form a complex network for expression control of symbiotic and accessory genes in the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum. A genome-wide survey of regulatory interactions was made possible with the design of a custom-made gene chip. Here, we report the first use of the microarray in a comprehensive and complete characterization of the B. japonicum NifA+sigma(54) regulon which forms an important node in the entire network. Comparative transcript profiles of anaerobically grown wild-type, nifA, and rpoN (1/2) mutant cells were complemented with a position-specific frequency matrix-based search for NifA- and sigma(54)-binding sites plus a simple operon definition. One of the newly identified NifA+sigma(54)-dependent genes, fdxN, encodes a ferredoxin required for efficient symbiotic nitrogen fixation, which makes it a candidate for being a direct electron donor to nitrogenase. The fdxN gene has an unconventional, albeit functional sigma(54 )promoter with the dinucleotide GA instead of the consensus GC motif at position -12. A GC-containing mutant promoter and the atypical GA-containing promoter of the wild type were disparately activated. Expression analyses were also carried out with two other NifA+sigma(54) targets (ectC; ahpC). Incidentally, the tiling-like design of the microarray has helped to arrive at completely revised annotations of the ectC- and ahpC-upstream DNA regions, which are now compatible with promoter locations. Taken together, the approaches used here led to a substantial expansion of the NifA+sigma(54 )regulon size, culminating in a total of 65 genes for nitrogen fixation and diverse other processes.

Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont, possesses a heme uptake system encoded by the gene cluster hmuVUT-hmuR-exbBD-tonB. Transcription of the divergently oriented hmuT and hmuR genes was previously found to be induced by iron limitation and to depend on a 21-bp promoter-upstream iron control element (ICE). Here, we show by deletion analysis that the full-length ICE is needed for this type of positive control. Additional genes associated with ICE-like motifs were identified in the B. japonicum genome, of which bll6680 and blr7895 code for bacterioferritin and rubrerythrin homologs, respectively. Transcription start site mapping revealed that their ICEs directly overlap with either the ؊10 promoter region or the transcription initiation site, suggesting an involvement of the ICE in negative control of both genes. Consistent with this inference was the observed downregulation of both genes under iron limitation, which in the case of bll6680 was shown to require an intact ICE motif. Using a yeast one-hybrid system, we demonstrated in vivo interaction of the iron response regulator (Irr) with all three ICEs. Moreover, specific in vitro binding of purified Irr protein to the ICE motifs of bll6680 and blr7895 was shown in electrophoretic mobility shift experiments. A genome-wide survey for iron-regulated genes with a custom-made Affymetrix gene chip revealed 17 genes to be induced and 68 to be repressed under iron-replete conditions. Remarkably, ICE-like motifs are associated with a large subset of those B. japonicum genes. We propose the ICE as an important cis-acting element in B. japonicum which represents the DNA-binding site for the Irr protein and, depending on its location within promoter regions, is involved in positive or negative control of the associated iron-regulated genes.