Joseph T. Wade

The transcription factor Ifh1 is a key regulator of yeast ribosomal protein genes.

Ribosomal protein (RP) genes in eukaryotes are coordinately regulated in response to growth stimuli and environmental stress, thereby permitting cells to adjust ribosome number and overall protein synthetic capacity to physiological conditions. Approximately 50% of RNA polymerase II transcription is devoted to RP genes. The transcriptional regulator Rap1 binds most yeast RP promoters, and Rap1 sites are important for coordinate regulation of RP genes. However, Rap1 is not the specific regulator that controls RP transcription because it also functions as a repressor, and many Rap1-activated promoters are not coordinately regulated with RP promoters. Here we show that the transcription factors Fhl1 and Ifh1 associate almost exclusively with RP promoters; association depends on Rap1 and (to a lesser extent) a DNA element at many RP promoters. Ifh1 is recruited to promoters via the forkhead-associated (FHA) domain of Fhl1; the level of Ifh1 associated with RP promoters determines the level of transcription; and environmental stress causes a marked reduction in the association of Ifh1, but not Fhl1 or Rap1. Thus, Ifh1 association with promoters is the key regulatory step for coordinate expression of RP genes.

Extensive functional overlap between σ factors in Escherichia coli

Bacterial core RNA polymerase (RNAP) must associate with a σ factor to recognize promoter sequences. Escherichia coli encodes seven σ factors, each believed to be specific for a largely distinct subset of promoters. Using microarrays representing the entire E. coli genome, we identify 87 in vivo targets of σ32, the heat-shock σ factor, and estimate that there are 120–150 σ32 promoters in total. Unexpectedly, 25% of these σ32 targets are located within coding regions, suggesting novel regulatory roles for σ32. The majority of σ32 promoter targets overlap with those of σ70, the housekeeping σ factor. Furthermore, their DNA sequence motifs are often interdigitated, with RNAPσ70 and RNAPσ32 initiating transcription in vitro with similar efficiency and from identical positions. σE-regulated promoters also overlap extensively with those for σ70. These results suggest that extensive functional overlap between σ factors is an important phenomenon.

Widespread Antisense Transcription in Escherichia coli

ABSTRACT The vast majority of annotated transcripts in bacteria are mRNAs. Here we identify ~1,000 antisense transcripts in the model bacterium Escherichia coli. We propose that these transcripts are generated by promiscuous transcription initiation within genes and that many of them regulate expression of the overlapping gene. IMPORTANCE The vast majority of known genes in bacteria are protein coding, and there are very few known antisense transcripts within these genes, i.e., RNAs that are encoded opposite the gene. Here we demonstrate the existence of ~1,000 antisense RNAs in the model bacterium Escherichia coli. Given the high potential for these RNAs to base pair with mRNA of the overlapping gene and the likelihood of clashes between transcription complexes of antisense and sense transcripts, we propose that antisense RNAs represent an important but overlooked class of regulatory molecule. The vast majority of known genes in bacteria are protein coding, and there are very few known antisense transcripts within these genes, i.e., RNAs that are encoded opposite the gene. Here we demonstrate the existence of ~1,000 antisense RNAs in the model bacterium Escherichia coli. Given the high potential for these RNAs to base pair with mRNA of the overlapping gene and the likelihood of clashes between transcription complexes of antisense and sense transcripts, we propose that antisense RNAs represent an important but overlooked class of regulatory molecule.

An allosteric mechanism of Rho-dependent transcription termination

Rho is the essential RNA helicase that sets the borders between transcription units and adjusts transcriptional yield to translational needs in bacteria. Although Rho was the first termination factor to be discovered, the actual mechanism by which it reaches and disrupts the elongation complex (EC) is unknown. Here we show that the termination-committed Rho molecule associates with RNA polymerase (RNAP) throughout the transcription cycle; that is, it does not require the nascent transcript for initial binding. Moreover, the formation of the RNAP–Rho complex is crucial for termination. We show further that Rho-dependent termination is a two-step process that involves rapid EC inactivation (trap) and a relatively slow dissociation. Inactivation is the critical rate-limiting step that establishes the position of the termination site. The trap mechanism depends on the allosterically induced rearrangement of the RNAP catalytic centre by means of the evolutionarily conserved mobile trigger-loop domain, which is also required for EC dissociation. The key structural and functional similarities, which we found between Rho-dependent and intrinsic (Rho-independent) termination pathways, argue that the allosteric mechanism of termination is general and likely to be preserved for all cellular RNAPs throughout evolution.

An HMG Protein, Hmo1, Associates with Promoters of Many Ribosomal Protein Genes and throughout the rRNA Gene Locus in Saccharomyces cerevisiae

ABSTRACT HMG proteins are architectural proteins that bind to DNA with low sequence specificity, but little is known about their genomic location and biological functions. Saccharomyces cerevisiae encodes 10 HMG proteins, including Hmo1, which is important for maximal transcription of rRNA. Here we use chromatin immunoprecipitation coupled with microarray analysis to determine the genome-wide association of Hmo1. Unexpectedly, Hmo1 binds strongly to the promoters of most ribosomal protein (RP) genes and to a number of other specific genomic locations. Hmo1 binding to RP promoters requires Rap1 and (to a lesser extent) Fhl1, proteins that also associate with RP promoters. Hmo1, like Fhl1 and Ifh1, typically associates with an IFHL motif in RP promoters, but deletion of the IFHL motif has a very modest effect on Hmo1 binding. Surprisingly, loss of Hmo1 abolishes binding of Fhl1 and Ifh1 to RP promoters but does not significantly affect the level of transcriptional activity. These results suggest that Hmo1 is required for the assembly of transcription factor complexes containing Fhl1 and Ifh1 at RP promoters and that proteins other than Fhl1 and Ifh1 also play an important role in RP transcription. Lastly, like mammalian UBF, Hmo1 associates at many locations throughout the rRNA gene locus, and it is important for processing of rRNA in addition to its role in rRNA transcription. We speculate that Hmo1 has a role in coordinating the transcription of rRNA and RP genes.

Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays

Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.

Genomic analysis of protein-DNA interactions in bacteria : insights into transcription and chromosome organization

Chromatin immunoprecipitation (ChIP) is a powerful method to measure protein–DNA interactions in vivo, and it can be applied on a genomic scale with microarray technology (ChIP‐chip). ChIP‐chip has been used extensively to map DNA–protein interactions across eukaryotic chromosomes. Here we review recent applications of ChIP‐chip to the study of bacteria, which provide important and unexpected insights into transcription and chromosome organization.

The transition from transcriptional initiation to elongation.

Transcription is the first step in gene expression, and its regulation underlies multicellular development and the response to environmental changes. Most studies of transcriptional regulation have focused on the recruitment of RNA polymerase to promoters. However, recent work has shown that, for many promoters, post-recruitment steps in transcriptional initiation are likely to be rate limiting. The rate at which RNA polymerase transitions from transcriptional initiation to elongation varies dramatically between promoters and between organisms and is the target of multiple regulatory proteins that can function to both repress and activate transcription.

Widespread suppression of intragenic transcription initiation by H-NS

Widespread intragenic transcription initiation has been observed in many species. Here we show that the Escherichia coli ehxCABD operon contains numerous intragenic promoters in both sense and antisense orientations. Transcription from these promoters is silenced by the histone-like nucleoid structuring (H-NS) protein. On a genome-wide scale, we show that 46% of H-NS-suppressed transcripts in E. coli are intragenic in origin. Furthermore, many intergenic promoters repressed by H-NS are for noncoding RNAs (ncRNAs). Thus, a major overlooked function of H-NS is to prevent transcription of spurious RNA. Our data provide a molecular description for the toxicity of horizontally acquired DNA and explain how this is counteracted by H-NS.

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5’ untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5’ end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5’ ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5’ UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.