Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor 32 rise greatly, resulting in the increased formation of the 32 holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on 32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving 32 expression from one plasmid and a ␤-galactosidase reporter gene driven by the 32 -dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on 32 activity. It was found that the recognition of the ؊35 region involves similar amino acid residues in regions 4.2 of E. coli 32 and 70 . Three conserved amino acids in region 2.3 of 32 were found to be only marginally important in determining activity in vivo. Differences between 32 and 70 in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.The master regulator for the heat shock response in Escherichia coli, now usually referred to as 32 , was identified as a sigma factor over 20 years ago (11,18). When E. coli cells are exposed to high temperatures (e.g., 42°C), the levels of 32 first rise steeply by a variety of different mechanisms and then level off at about twice the initial level (6,10,25). 32 binds to core RNA polymerase (RNAP) and directs the RNAP to the heat shock promoters, for which the consensus sequence differs from those utilized by RNAP containing the housekeeping sigma factor, 70 (4, 10, 35, 36, 38). The difference is pronounced in the Ϫ10 region, but in the Ϫ35 region the two classes of promoters have a 4-base-pair sequence in common. In view of the homology between the two proteins in region 4.2 (see Fig. 1A), it had been postulated early on that the recognition of the Ϫ35 regions would involve similar amino acids of 32 and 70 (30). While the most evident role of 32 is in directing the expression of genes allowing the cell to deal with the consequences of heat shock, it is an essential protein at physiological (37°C) temperatures (37). Despite its importance to the survival of the cell, few studies have addressed the roles of particular amino acids. Amino acids in region 2.1 were found to markedly affect the stability of 32 in vivo (12); a stretch of amino acids between regions 2 and 3 of 32 is known to participate in the binding of DnaK (21), which functions as an anti-sigma factor of 32 ; and amino acids that play a role in the interaction of 32 with the core have been identified (13,14). In addition, random linker insertion mutagenesis has provided information concerning the roles of various regions of 32 in determining its activity (21). Here we report the results of targeted mutagenesis of amino acids in variou...

5S rRNAs are ubiquitous components of prokaryotic, chloroplast, and eukaryotic cytosolic ribosomes but are apparently absent from mitochondrial ribosomes (mitoribosomes) of many eukaryotic groups including animals and fungi. Nevertheless, a clearly identifiable, mitochondrion-encoded 5S rRNA is present in Acanthamoeba castellanii, a member of Amoebozoa. During a search for additional mitochondrial 5S rRNAs, we detected small abundant RNAs in other members of Amoebozoa, namely, in the lobose amoeba Hartmannella vermiformis and in the myxomycete slime mold Physarum polycephalum. These RNAs are encoded by mitochondrial DNA (mtDNA), cosediment with mitoribosomes in glycerol gradients, and can be folded into a secondary structure similar to that of bona fide 5S rRNAs. Further, in the mtDNA of another slime mold, Didymium nigripes, we identified a region that in sequence, potential secondary structure, and genomic location is similar to the corresponding region encoding the Physarum small RNA. A mtDNA-encoded small RNA previously identified in Dictyostelium discoideum is here shown to share several characteristics with known 5S rRNAs. Again, we detected genes encoding potential homologs of this RNA in the mtDNA of three other species of the genus Dictyostelium as well as in a related genus, Polysphondylium. Taken together, our results indicate a widespread occurrence of small, abundant, mtDNA-encoded RNAs with 5S rRNA-like structures that are associated with the mitoribosome in various amoebozoan taxa. Our working hypothesis is that these novel small abundant RNAs represent radically divergent mitochondrial 5S rRNA homologs. We posit that currently unrecognized 5S-like RNAs may exist in other mitochondrial systems in which a conventional 5S rRNA cannot be identified.

The heat shock sigma factor ( 32 in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of 32 is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, 70 . This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha-and deltaproteobacteria, it is a tryptophan. In vitro the alaninefor-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing 32 , implying that H107 plays a role in formation or maintenance of the strandseparated complex. In vivo, the H107A substitution in 32 impedes recovery from heat shock (exposure to 42°C), and it also leads to overexpression at lower temperatures (30°C) of the Flu protein, which is associated with biofilm formation.

Global gene expression analysis using Affymetrix GeneChip Human Transcriptome Array (HTA) 2.0 combined with Affymetrix Transcriptome Analysis Console (TAC) Software empowers scientists not only to gain information about RNA expression at the gene‐level, but also about RNA alternative splicing at the exon‐level. Real‐time PCR is routinely used to verify microarray results due to its high sensitivity and wide dynamic range, and was used in this study to validate Affymetrix GeneChip HTA 2.0 data. Gene‐level expression data for the classic MAQC A and MAQC B samples (used by the MicroArray Quality Control Consortium) were validated with commercially available primers and design software. Excellent fold‐change correlation of microarray and qPCR data were observed using USB VeriQuest Probe real‐time PCR (R=0.96, slope=0.70). For alternative splicing validation of Affymetrix GeneChip HTA 2.0, total RNA from three different tissues (liver, muscle and brain) were used. Exon level information provided by TAC Software was utilized to design custom PCR primers for USB VeriQuest SYBR Green real‐time PCR. All alternative splicing events selected for validation were confirmed by real‐time PCR. We show excellent inter‐platform concordance between Affymetrix microarray and VeriQuest real‐time PCR data. Grant Funding Source: Supported by Affymetrix, Inc.

Formalin-fixed and paraffin-embedded (FFPE) tissue samples represent a largely untapped wealth of information on the transcriptional states of clinically archived materials for retrospective studies. Although the RNA is degraded during fixation and storage and has been considered unusable, Affymetrix has developed SensationPlus™, a target-preparation method that allows transcriptional profiling from 20 ng of FFPE total RNA using GeneChip™ arrays. We will demonstrate that SensationPlus™ expression profiles from decade old FFPE materials are extremely similar to data from their matched fresh frozen (FF) RNA counterparts. Citation Format: Sangpen Chamnongpol, Andrea Ujvari, Olga Kourennaia, Kim Myers, Lisa Bowers, Jesse Fisher, Marc Post, Chris Kubu. Differential gene expression profiles of FFPE samples with SensationPlus™ and GeneChip™ arrays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1797. doi:10.1158/1538-7445.AM2013-1797