RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates

Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires σ factor that participates in both promoter recognition and DNA melting. RNAP lacking σ (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions. We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various σ factors. Using bubble templates containing the σD-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of σ. This supports a model in which the UP element acts primarily through the α subunit of core enzyme to increase the initial association of RNAP with the promoter. Core and holoenzyme do differ substantially in the template positions chosen for initiation: σD restricts initiation to sites 8–9 nucleotides downstream of the conserved −10 element. Remarkably, σA also has a dramatic effect on start-site selection even though the σA holoenzyme is inactive on the corresponding homoduplexes. The start sites chosen by the σA holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved −10 hexamer recognized by σA. Thus, σA appears to recognize the −10 region even in a single-stranded state. We propose that in addition to its described roles in promoter recognition and start-site melting, σ also localizes the transcription start site.

The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization

Positioning of the Z ring at the midcell site in Escherichia coli is assured by the min system, which masks polar sites through topological regulation of MinC, an inhibitor of division. To study how MinC inhibits division, we have generated a MalE-MinC fusion that retains full biological activity. We find that MalE-MinC interacts with FtsZ and prevents polymerization without inhibiting FtsZ's GTPase activity. MalE-MinC19 has reduced ability to inhibit division, reduced affinity for FtsZ, and reduced ability to inhibit FtsZ polymerization. These results, along with MinC localization, suggest that MinC rapidly oscillates between the poles of the cell to destabilize FtsZ filaments that have formed before they mature into polar Z rings.

Modulation of 5' splice site choice in pre-messenger RNA by two distinct steps.

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). Here, we perturbed the U1 snRNP in HeLa cell nuclear extract by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for 5' splice site selection. Accordingly, we found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. Our results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection.

Functional association between promoter structure and transcript alternative splicing

It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription.

The Saccharomyces cerevisiae Homologue of Human Wiskott–Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex

Yeast Las17 protein is homologous to the Wiskott–Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Δ ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Δ cells showed that receptor-mediated internalization of α factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.

The Interaction and Colocalization of Sam68 with the Splicing-associated Factor YT521-B in Nuclear Dots Is Regulated by the Src Family Kinase p59fyn

Alternative pre-mRNA splicing patterns can change an extracellular stimulus, but the signaling pathways leading to these changes are still poorly characterized. Here, we describe a tyrosine-phosphorylated nuclear protein, YT521-B, and show that it interacts with the nuclear transcriptosomal component scaffold attachment factor B, and the 68-kDa Src substrate associated during mitosis, Sam68. Northern blot analysis demonstrated ubiquitous expression, but detailed RNA in situ analysis revealed cell type specificity in the brain. YT521-B protein is localized in the nucleoplasm and concentrated in 5–20 large nuclear dots. Deletion analysis demonstrated that the formation of these dots depends on the presence of the amino-terminal glutamic acid-rich domain and the carboxyl-terminal glutamic acid/arginine-rich region. We show that the latter comprises an important protein–protein interaction domain. The Src family kinase p59fyn-mediated tyrosine phosphorylation of Sam68 negatively regulates its association with YT521-B, and overexpression of p59fyn dissolves nuclear dots containing YT521-B. In vivo splicing assays demonstrated that YT521-B modulates alternative splice site selection in a concentration-dependent manner. Together, our data indicate that YT521-B and Sam68 may be part of a signal transduction pathway that influences splice site selection.

Tn5/IS50 target recognition

This communication reports an analysis of Tn5/IS50 target site selection by using an extensive collection of Tn5 and IS50 insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from in vitro and in vivo transposition events. Since the data sets are consistent and transposase was the only protein present in vitro, this demonstrates that target selection is a property of only transposase. There appear to be two factors governing target selection. A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn5/IS50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn5/IS50 target site is A-GNTYWRANC-T. However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion. This suggests that Tn5/IS50 target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too. Synthetic target sequences were designed and used to test and confirm this model.

DNA binding sites for the Mlc and NagC proteins: regulation of nagE, encoding the N-acetylglucosamine-specific transporter in Escherichia coli

The NagC and Mlc proteins are homologous transcriptional regulators that control the expression of several phosphotransferase system (PTS) genes in Escherichia coli. NagC represses nagE, encoding the N-acetylglucosamine-specific transporter, while Mlc represses three PTS operons, ptsG, manXYZ and ptsHIcrr, involved in the uptake of glucose. NagC and Mlc can bind to each others operator, at least in vitro. A binding site selection procedure was used to try to distinguish NagC and Mlc sites. The major difference was that all selected NagC binding sites had a G or a C at positions +11/–11 from the centre of symmetry. This is also the case for most native NagC sites, but not the nagE operator, which thus looks like a potential Mlc target. The nagE operator does exhibit a higher affinity for Mlc than NagC, but no regulation of nagE by physiological concentrations of Mlc was detected in vivo. Regulation of wild-type nagE by NagC is achieved because of the chelation effect due to a second high affinity NagC operator covering the nagB promoter. Replacing the A/T at +11/–11 with C/G allows repression by NagC in the absence of the nagB operator.

Fully modified 2′ MOE oligonucleotides redirect polyadenylation

Many genes have been described and characterized that have alternative polyadenylation signals at the 3′-end of their pre-mRNAs. Many of these same messages also contain destabilization motifs responsible for rapid degradation of the mRNA. Polyadenylation site selection can thus determine the stability of an mRNA. Fully modified 2′-O-methoxy ethyl/phosphorothioate oligonucleotides that hybridize to the 3′-most polyadenylation site or signal of E-selectin were able to inhibit polyadenylation at this site and redirect it to one of two upstream cryptic sites. The shorter transcripts produced after antisense treatment have fewer destabilization sequences, increased mRNA stability and altered protein expression. This study demonstrates that antisense oligonucleotides can be successfully employed to redirect polyadenylation. This is the first demonstration of the use of oligonucleotides to increase, rather than decrease, abundance of a message.

Heterogeneity in polyadenylation cleavage sites in mammalian mRNA sequences: implications for SAGE analysis

The analysis of a human thyroid serial analysis of gene expression (SAGE) library shows the presence of an abundant SAGE tag corresponding to the mRNA of thyroglobulin (TG). Additional, less abundant tags are present that can not be linked to any other known gene, but show considerable homology to the wild-type TG tag. To determine whether these tags represent TG mRNA molecules with alternative cleavage, 3′-RACE clones were sequenced. The results show that the three putative TG SAGE tags can be attributed to TG transcripts and reflect the use of alternative polyadenylation cleavage sites downstream of a single polyadenylation signal in vivo. By screening more than 300 000 sequences corresponding to human, mouse and rat transcripts for this phenomenon we show that a considerable percentage of mRNA transcripts (44% human, 22% mouse and 22% rat) show cleavage site heterogeneity. When analyzing SAGE-generated expression data, this phenomenon should be considered, since, according to our calculations, 2.8% of human transcripts show two or more different SAGE tags corresponding to a single gene because of alternative cleavage site selection. Both experimental and in silico data show that the selection of the specific cleavage site for poly(A) addition using a given polyadenylation signal is more variable than was previously thought.

RNA–protein hybrid ribozymes that efficiently cleave any mRNA independently of the structure of the target RNA

Ribozyme activity in vivo depends on achieving high-level expression, intracellular stability, target colocalization, and cleavage site access. At present, target site selection is problematic because of unforeseeable secondary and tertiary RNA structures that prevent cleavage. To overcome this design obstacle, we wished to engineer a ribozyme that could access any chosen site. To create this ribozyme, the constitutive transport element (CTE), an RNA motif that has the ability to interact with intracellular RNA helicases, was attached to our ribozymes so that the helicase-bound, hybrid ribozymes would be produced in cells. This modification significantly enhanced ribozyme activity in vivo, permitting cleavage of sites previously found to be inaccessible. To confer cleavage enhancement, the CTE must retain helicase-binding activity. Binding experiments demonstrated the likely involvement of RNA helicase(s). We found that attachment of the RNA motif to our tRNA ribozymes leads to cleavage in vivo at the chosen target site regardless of the local RNA secondary or tertiary structure.

Conservation and diversification in homeodomain-DNA interactions: a comparative genetic analysis.

Nearly all metazoan homeodomains (HDs) possess DNA binding targets that are related by the presence of a TAAT sequence. We use an in vitro genetic DNA binding site selection assay to refine our understanding of the amino acid determinants for the recognition of the TAAT site. Superimposed upon the conserved ability of metazoan HDs to recognize a TAAT core is a difference in their preference for the bases that lie immediately 3' to it. Amino acid position 50 of the HD has been shown to discriminate among these base pairs, and structural studies have suggested that water-mediated hydrogen bonds and van der Waals contacts underlie for this ability. Here, we show that each of six amino acids tested at position 50 can confer a distinct DNA binding specificity.

Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements.

The Drosophila CF2II protein, which contains zinc fingers of the Cys2His2 type and recognizes an A+T-rich sequence, behaves in cell culture as an activator of a reporter chloramphenicol acetyltransferase gene. This activity depends on C-terminal but not N-terminal zinc fingers, as does in vitro DNA binding. By site-specific mutagenesis and binding site selection, we define the critical amino acid-base interactions. Mutations of single amino acid residues at the leading edge of the recognition helix are rarely neutral: many result in a slight change in affinity for the ideal DNA target site; some cause major loss of affinity; and others change specificity for as many as two bases in the target site. Compared to zinc fingers that recognize G+C-rich DNA, CF2II fingers appear to bind to A+T-rich DNA in a generally similar manner, but with additional flexibility and amino acid-base interactions. The results illustrate how zinc fingers may be evolving to recognize an unusually diverse set of DNA sequences.

Enhancer-dependent interaction between 5' and 3' splice sites in trans.

Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing enhancers that act by promoting the activity of upstream splice sites. Here we show that RNA molecules containing a 3' splice site and enhancer sequence are efficiently spliced in trans to RNA molecules containing normally cis-spliced 5' splice sites or to normally trans-spliced spliced leader RNAs from lower eukaryotes. In addition, we show that this reaction is stimulated by (Ser + Arg)-rich splicing factors that are known to promote protein-protein interactions in the cis-splicing reaction. Thus, splicing enhancers facilitate the assembly of protein complexes on RNAs containing a 3' splice site, and this complex is sufficiently stable to functionally interact with 5' splice sites located on separate RNAs. This trans-splicing is mediated by interactions between (Ser + Arg)-rich splicing factors bound to the enhancer and general splicing factors bound to the 5' and 3' splice sites. These same interactions are likely to play a crucial role in alternative splicing and splice-site selection in cis.

Identification of the gene (SSU71/TFG1) encoding the largest subunit of transcription factor TFIIF as a suppressor of a TFIIB mutation in Saccharomyces cerevisiae.

Mutations in the Saccharomyces cerevisiae SSU71 gene were isolated as suppressors of a transcription factor TFIIB defect that confers both a cold-sensitive growth defect and a downstream shift in transcription start-site selection at the cyc1 locus. The ssu71-1 suppressor not only suppresses the conditional phenotype but also restores the normal pattern of transcription initiation at cyc1. In addition, the ssu71-1 suppressor confers a heat-sensitive phenotype that is dependent upon the presence of the defective form of TFIIB. Molecular and genetic analysis of the cloned SSU71 gene demonstrated that SSU71 is a single-copy essential gene encoding a highly charged protein with a molecular mass of 82,194 daltons. Comparison of the deduced Ssu71 amino acid sequence with the protein data banks revealed significant similarity to RAP74, the larger subunit of the human general transcription factor TFIIF. Moreover, Ssu71 is identical to p105, a component of yeast TFIIF. Taken together, these data demonstrate a functional interaction between TFIIB and the large subunit of TFIIF and that this interaction can affect start-site selection in vivo.