Upstream Poly Research Articles

Splicing factor Prp18p promotes genome-wide fidelity of consensus 3'-splice sites.

The fidelity of splice site selection is critical for proper gene expression. In particular, proper recognition of 3'-splice site (3'SS) sequences by the spliceosome is challenging considering the low complexity of the 3'SS consensus sequence YAG. Here, we show that absence of the Prp18p splicing factor results in genome-wide activation of alternative 3'SS in S. cerevisiae, including highly unusual non-YAG sequences. Usage of these non-canonical 3'SS in the absence of Prp18p is enhanced by upstream poly(U) tracts and by their potential to interact with the first intronic nucleoside, allowing them to dock in the spliceosome active site instead of the normal 3'SS. The role of Prp18p in 3'SS fidelity is facilitated by interactions with Slu7p and Prp8p, but cannot be fulfilled by Slu7p, identifying a unique role for Prp18p in 3'SS fidelity. This fidelity function is synergized by the downstream proofreading activity of the Prp22p helicase, but is independent from another late splicing helicase, Prp43p. Our results show that spliceosomes exhibit remarkably relaxed 3'SS sequenceusage in the absence of Prp18p and identify a network of spliceosomal interactions centered on Prp18p which are required to promote the fidelity of the recognition of consensus 3'SS sequences.

Elongation rate of RNA polymerase II affects pausing patterns across 3′ UTRs

Yeast mRNAs are polyadenylated at multiple sites in their 3' untranslated regions (3' UTRs), and poly(A) site usage is regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II). Slow Pol II derivatives favor upstream poly(A) sites, and fast Pol II derivatives favor downstream poly(A) sites. Transcriptional elongation and polyadenylation are linked at the nucleotide level, presumably reflecting Pol II dwell time at each residue that influences the level of polyadenylation. Here, we investigate the effect of Pol II elongation rate on pausing patterns and the relationship between Pol II pause sites and poly(A) sites within 3' UTRs. Mutations that affect Pol II elongation rate alter sequence preferences at pause sites within 3' UTRs, and pausing preferences differ between 3' UTRs and coding regions. In addition, sequences immediately flanking the pause sites show preferences that are largely independent of Pol II speed. In wild-type cells, poly(A) sites are preferentially located< 50 nucleotides upstream from Pol II pause sites, but this spatial relationship is diminished in cells harboring Pol II speed mutants. Based on a random forest classifier, Pol II pause sites are modestly predicted by the distance to poly(A) sites but are better predicted by the chromatin landscape in Pol II speed derivatives. Transcriptional regulatory proteins can influence the relationship between Pol II pausing and polyadenylation but in a manner distinct from Pol II elongation rate derivatives. These results indicate a complex relationship between Pol II pausing and polyadenylation.

Mechanism and consequences of herpes simplex virus 1-mediated regulation of host mRNA alternative polyadenylation.

Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. HSV-1-induced mRNAs polyadenylated at intronic PAS (IPA) are exported into the cytoplasm while APA isoforms with extended 3’ UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Finally we provide evidence that HSV-induced IPA isoforms are translated. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host response that includes DoTT and changes in APA profiles.

Gene Editing Targeting the DUX4 Polyadenylation Signal: A Therapy for FSHD?

Facioscapulohumeral dystrophy (FSHD, OMIM: 158900, 158901) is the most common dystrophy in adults and so far, there is no treatment. Different loci of the disease have been characterized and they all lead to the aberrant expression of the DUX4 protein, which impairs the function of the muscle, ultimately leading to cell death. Here, we used gene editing to try to permanently shut down DUX4 expression by targeting its poly(A) sequence. We used transcription activator-like effector nucleases (TALEN) and CRISPR-Cas9 nucleases in vitro on FSHD myoblasts. More than 150 TOPO clones were sequenced and only indels were observed in 4%. Importantly, in 2 of them, the DUX4 poly(A) signal was eliminated at the genomic level but DUX4 mRNA was still produced thanks to the use of a non-canonical upstream poly(A) signal sequence. These experiments show that targeting DUX4 PAS at the genomic level might not be an appropriate gene editing strategy for FSHD therapy.

CCR4 and CAF1 deadenylases have an intrinsic activity to remove the post-poly(A) sequence.

MicroRNAs (miRNAs) recruit the CCR4–NOT complex, which contains two deadenylases, CCR4 and CAF1, to promote shortening of the poly(A) tail. Although both CCR4 and CAF1 generally have a strong preference for poly(A) RNA substrates, it has been reported from yeast to humans that they can also remove non-A residues in vitro to various degrees. However, it remains unknown how CCR4 and CAF1 remove non-A sequences. Herein we show that Drosophila miRNAs can promote the removal of 3′-terminal non-A residues in an exonucleolytic manner, but only if an upstream poly(A) sequence exists. This non-A removing reaction is directly catalyzed by both CCR4 and CAF1 and depends on the balance between the length of the internal poly(A) sequence and that of the downstream non-A sequence. These results suggest that the CCR4–NOT complex has an intrinsic activity to remove the 3′-terminal non-A modifications downstream from the poly(A) tail.

Abstract A48: Role of premature mRNA cleavage and alternative polyadenylation in driving breast cancer

Abstract There is increasing appreciation that alterations to epigenetic mechanisms contribute directly to breast cancer development. In cancer cells, one such mechanism, called alternative polyadenylation (APA), has been observed to cause a general shortening of mRNA through the usage and premature cleavage of upstream poly(A) signals (PAS). However, while it is hypothesized that APA may be linked to cancer pathogenesis, specific examples of causality between APA and cancer development have remained elusive. We report the identification of a truncated tumor suppressor, MAGI3, at both the mRNA and protein levels from the MDA-MB-231 breast cancer cell line. Using 3' rapid amplification of cDNA ends (RACE), we have determined that the truncated transcript arises due to premature cleavage and APA at a cryptic intronic PAS. We have previously demonstrated genetically and biochemically that MAGI3 functions as a mammary tumor suppressor by antagonizing the oncoprotein and Hippo pathway effector, YAP, via a physical sequestration mechanism. Importantly, here we find that the truncated gene product encoded by this aberrant MAGI3 transcript acts as a dominant-negative protein, preventing MAGI3-YAP physical association and thereby causing YAP nuclear activation and malignant transformation of cells. Since “epi-mutations” such as these play an important role in breast cancer development, yet cannot be detected by microarray or genome sequencing methodologies, we are currently undertaking analysis of large-scale, breast cancer RNA-Seq data to identify tumors that express the truncated driver mRNA and to determine the frequency of APA products in breast cancer. Knowledge of the clinically relevant proteins produced by APA that are unique to cancer cells may lead to new therapeutic opportunities for breast cancer. Citation Format: Thomas K. Ni, Charlotte Kuperwasser. Role of premature mRNA cleavage and alternative polyadenylation in driving breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A48.

Discrete lot sizing and scheduling problem under batch processing constraints in the semiconductor manufacturing

Highly advanced technology products are manufactured on the series of equipment having various characteristics and requirements. The deposition process of organic light emitting diode, for example, is performed in chambers for a long manufacturing time, while various product devices belonging to the same family can be processed in sequence before the preventive maintenance schedule or the refill of chemical gas required. The deposition process plays as a bottleneck, and its productivity of the schedule is critical to the upstream low-temperature poly silicon process and the downstream encapsulation and module processes as well. The batch family scheduling problem is formulated using the mixed-integer programming (MIP) in consideration of the family setups, lot sizing, supplementary mask tools requirements, and material exhaustion requirements. It is shown that the planning and scheduling decisions can be made simultaneously in an integrated model, and that it can be implemented in the actual manufacturing line. Through the optimized model analysis, the capacity can be enhanced by 20∼30 % without losing the throughput and demand satisfaction as well.

Immunoglobulin heavy chain gene regulation through polyadenylation and splicing competition

The immunoglobulin heavy chain (IgH) genes, which encode one of the two chains of antibody molecules, were the first cellular genes shown to undergo developmentally regulated alternative RNA processing. These genes produce two different mRNAs from a single primary transcript. One mRNA is cleaved and polyadenylated at an upstream poly(A) signal while the other mRNA removes this poly(A) signal by RNA splicing and is cleaved and polyadenylated at a downstream poly(A) site. A broad range of studies have been performed to understand the mechanism of IgH RNA processing regulation during B lymphocyte development. The model that has emerged is much more complex than envisioned by the earliest view of regulation through poly(A) signal choice. Regulation requires that the IgH gene contain competing splice and cleavage-polyadenylation reactions with balanced efficiencies. Because non-IgH genes with these structural features also can be regulated, IgH gene-specific sequence elements are not required for regulation. Changes in cleavage-polyadenylation and RNA splicing, as well as pol II elongation, all contribute to IgH developmental RNA processing regulation. Multiple factors are likely involved in the regulation during B lymphocyte maturation. Additional biologically relevant factors that contribute to IgH regulation remain to be identified and incorporated into a mechanistic model for regulation. Much of the work to date confirms the complex nature of IgH mRNA regulation and suggests that a thorough understanding of this control will remain a challenge. However, it is also likely that such understanding will help elucidate novel mechanisms of RNA processing regulation.

Outline of a Genome Navigation System Based on the Properties of GA-Sequences and Their Flanks

Introducing a new method to visualize large stretches of genomic DNA (see Appendix S1) the article reports that most GA-sequences [1] shared chains of tetra-GA-motifs and contained upstream poly(A)-segments. Although not integral parts of them, Alu-elements were found immediately upstream of all human and chimpanzee GA-sequences with an upstream poly(A)-segment. The article hypothesizes that genome navigation uses these properties of GA-sequences in the following way. (1) Poly(A) binding proteins interact with the upstream poly(A)-segments and arrange adjacent GA-sequences side-by-side (‘GA-ribbon’), while folding the intervening DNA sequences between them into loops (‘associated DNA-loops’). (2) Genome navigation uses the GA-ribbon as a search path for specific target genes that is up to 730-fold shorter than the full-length chromosome. (3) As to the specificity of the search, each molecule of a target protein is assumed to catalyze the formation of specific oligomers from a set of transcription factors that recognize tetra-GA-motifs. Their specific combinations of tetra-GA motifs are assumed to be present in the particular GA-sequence whose associated loop contains the gene for the target protein. As long as the target protein is abundant in the cell it produces sufficient numbers of such oligomers which bind to their specific GA-sequences and, thereby, inhibit locally the transcription of the target protein in the associated loop. However, if the amount of target protein drops below a certain threshold, the resultant reduction of specific oligomers leaves the corresponding GA-sequence ‘denuded’. In response, the associated DNA-loop releases its nucleosomes and allows transcription of the target protein to proceed. (4) The Alu-transcripts may help control the general background of protein synthesis proportional to the number of transcriptionally active associated loops, especially in stressed cells. (5) The model offers a new mechanism of co-regulation of protein synthesis based on the shared segments of different GA-sequences.

The poly(A) site sequence in HDV RNA alters both extent and rate of self-cleavage of the antigenomic ribozyme

The ribozyme self-cleavage site in the antigenomic sequence of hepatitis delta virus (HDV) RNA is 33-nt downstream of the poly(A) site for the delta antigen mRNA. An HDV antigenomic ribozyme precursor RNA that included the upstream poly(A) processing site was used to test the hypothesis that nonribozyme sequence near the poly(A) site could affect ribozyme activity. Relative to ribozyme precursor without the extra upstream sequences, the kinetic profile for self-cleavage of the longer precursor was altered in two ways. First, only half of the precursor RNA self-cleaved. The cleaved fraction could be increased or decreased with mutations in the upstream sequence. These mutations, which were predicted to alter the relative stability of competing secondary structures within the precursor, changed the distribution of alternative RNA structures that are resolved in native-gel electrophoresis. Second, the active fraction cleaved with an observed rate constant that was higher than that of the ribozyme without the upstream sequences. Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1–2 mM). Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.

In vivo evidence that defects in the transcriptional elongation factors RPB2, TFIIS, and SPT5 enhance upstream poly(A) site utilization.

While a number of proteins are involved in elongation processes, the mechanism for action of most of these factors remains unclear primarily because of the lack of suitable in vivo model systems. We identified in yeast several genes that contain internal poly(A) sites whose full-length mRNA formation is reduced by mutations in RNA polymerase II subunit RPB2, elongation factor SPT5, or TFIIS. RPB2 and SPT5 defects also promoted the utilization of upstream poly(A) sites for genes that contain multiple 3' poly(A) signaling sequences, supporting a role for elongation in differential poly(A) site choice. Our data suggest that elongation defects cause increased transcriptional pausing or arrest that results in increased utilization of internal or upstream poly(A) sites. Transcriptional pausing or arrest can therefore be visualized in vivo if a gene contains internal poly(A) sites, allowing biochemical and genetic study of the elongation process.

3' nontranslated RNA signals required for replication of hepatitis C virus RNA.

We describe a mutational analysis of the 3' nontranslated RNA (3'NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3'NTR, namely, the highly conserved 3' terminal 98-nucleotide (nt) segment (3'X), an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5' end of the 3'NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3'X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3' end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3'-most 150 nt of the HCV-N genome [the 3'X region and the 3' 52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3'NTR plays a facilitating role in replication but is not absolutely required.

Design and Structural Analysis of Hairpin-TFO for Transcriptional Activation of Genes in S. cerevisiae

Triplex forming oligonucleotides (TFOs) have the potential to modulate gene expression. While most of the experiments are directed towards triplex mediated inhibition of gene expression the strategy potentially could be used for gene specific activation. In an attempt to design a strategy for gene specific activation in vivo applicable to a large number of genes we have designed a TFO based activator-target system which may be utilized in Saccharomyces cerevisiae or any other system where Ga14 protein is ectopically expressed. The total genome sequence of Saccharomyces cerevisiae and expression profiles were used to select the target genes with upstream poly (pu/py) sequences. We have utilized the paradigm of Gal4 protein and its binding site. We describe here the selection of target genes and design of hairpin-TFO including the targeting sequences containing polypurine stretch found in the upstream promoter regions of weakly expressed genes. We demonstrate, the formation of hairpin-TFO, its binding to Gal4 protein, its ability to form triplex with the target duplex in vitro, the effect of polyethylenimine on complex formation and discuss the implication on in vivo transcription activation.

An RNA polymerase pause site is associated with the immunoglobulin mus poly(A) site.

Immunoglobulin mu alternative RNA processing is regulated during B-cell maturation and requires balanced efficiencies of the competing splice (mum) and cleavage-polyadenylation (mus) reactions. When we deleted sequences 50 to 200 nucleotides beyond the mus poly(A) site, the mus/mum mRNA ratio decreased three- to eightfold in B, plasma, and nonlymphoid cells. The activity could not be localized to a smaller fragment but did function in heterologous contexts. Our data suggest that this region contains an RNA polymerase II pause site that enhances the use of the mus poly(A) site. First, known pause sites replaced the activity of the deleted fragment. Second, the mu fragment, when placed between tandem poly(A) sites, enhanced the use of the upstream poly(A) site. Finally, nuclear run-ons detected an increase in RNA polymerase loading just downstream from the mus poly(A) site, even when the poly(A) site was inactivated. When this mu fragment and another pause site were inserted 1 kb downstream from the mus poly(A) site, they no longer affected the mRNA expression ratio, suggesting that pause sites affect poly(A) site use over a limited distance. Fragments from the immunoglobulin A gene were also found to have RNA polymerase pause site activity.

Early polyadenylation signals of human papillomavirus type 31 negatively regulate capsid gene expression.

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed upon keratinocyte differentiation from a promoter located in the E7 open reading frame (ORF) of the early region. Late transcripts must therefore pass through and ignore the early polyadenylation sequences to use the downstream late AAUAAA element located at the end of the L1 ORF. To identify sequences which modulate downstream capsid gene expression, a variety of substitution mutations were introduced into the early polyadenylation signal and studied first in the context of polycistronic luciferase reporter constructs. Removal of the G/U-rich cleavage stimulation factor (CstF) binding sites and the degenerate cleavage and polyadenylation specificity factor binding sites, UAUAUA, had minimal effect on downstream expression as defined by luciferase activities. This is in contrast to the deletion of the HPV-31 early AAUAAA element, which resulted in a dramatic increase in downstream expression. Additional sequences within the first 800 bp of the L2 ORF were also found to negatively regulate capsid expression in luciferase assays. To determine how these mutations influence gene expression in the context of the complete HPV-31 genome, recombinant genomes were constructed that contained a substitution in the AAUAAA sequence, an inserted strong CstF binding site, an inserted simian virus 40 (SV40) late poly(A) signal, or a substitution of the 5'-most 800 nucleotides of the L2 ORF. Reductions in both transient and stable replication were observed with the recombinant genomes containing the strong CstF site or the late SV40 signal, suggesting that alterations in the strength of the upstream poly(A) signal influence expression of viral replication factors. Similarly, disruption of the L2 ORF resulted in a significant reduction in genome replication and an inability to be maintained stably. In contrast, genomes containing a substitution of the AAUAAA sequence had increased levels of transient and stable replication. Quantitation of late transcripts following keratinocyte differentiation in methylcellulose also showed a reduction in downstream capsid gene expression in lines containing genomes with the strong CstF site or the late SV40 signal mutations, while a significant increase in expression was detected in the lines with genomes lacking the AAUAAA sequence. These studies demonstrate that capsid gene expression in HPV-31 requires an inefficient early poly(A) signal which is defined primarily by the AAUAAA element as well as a major negative regulatory element located within the L2 ORF.

Kinetics of mRNA polyadenylation and its relation to transcription termination in eukaryotes

Processing of most eukaryotic mRNAs includes and polyadenylation of the nascent transcript. Until now there has been no method for the reliable measurement of these processes in vivo. Therefore, in the present work a new technique was developed for measuring precisely the rate of cleavage and polyadenylation in vivo. The method uses a cis-antisense element targeted to an upstream poly (A) signal. Duplex formation of the antisense element with the poly (A) signal region prevents polyadenylation. In a series of expression vector constructs the antisense element was moved increasing distances downstream of its target poly (A) site, reasoning that if it takes the polymerase longer to reach the antisense element, polyadenylation would have more time to occur. Using this method the half time for commitment to cleavage and polyadenylation at the SV40 early poly (A) site and SPA (synthetic poly A site) was found to be 5 seconds. It was found that strong sites (SV 40 late poly (A) site) were processed faster. Commitment to cleavage and polyadenylation was found to be a multistep process. The expression results were confirmed with the help of RNase protection assay. Relationship between polyadenylation and transcription termination was also studied by using G-free assay and found to be positively correlated. Present data support looping moded suggesting some communication exists between polyadenylation complex and RNA pol. II for transcription termination.

Different positioning elements select poly(A) sites at the 3'-end of GCN4 mRNA in the yeast Saccharomyces cerevisiae.

Cleavage and polyadenylation of eukaryotic mRNA requires efficiency and positioning elements in the 3'-untranslated region (3'-UTR) of the mRNA. Specific point mutations were introduced into the yeast GCN4 3'-UTR to detect sequence motifs which are involved in the positioning of the poly(A) site. 3'-End proces-sing activities of different GCN4 3'-UTR alleles were measured in an in vivo test system. Point mutations in an AAGAA motif defocussed selection of the poly(A) sites of the GCN4 3'-UTR to various additional poly(A) sites instead of the single site of the wild-type GCN4 3'-UTR. A strain with an intact wild-type GCN4 3'-UTR but impaired in RNA15 encoding an RNA-binding processing factor showed a similar defocussed pattern of poly(A) site selection. Remarkably, two additional sequence motifs upstream of the AAGAA motif which resemble yeast efficiency motifs independently affected poly(A) site positioning but not efficiency of 3'-end processing. Mutations in one motif resulted in an additional upstream poly(A) site. Alterations of the other motif shifted the poly(A) sites exclusively to two downstream poly(A) sites. These data suggest several contact points between the precursor mRNA and the polyadenylation machinery in yeast.

Specific Transcriptional Pausing Activates Polyadenylation in a Coupled In Vitro System

We have developed a coupled in vitro transcription–polyadenylation system to investigate RNA polymerase II (Pol II) termination, which depends on active polyadenylation of the nascent RNA. Specific G-rich sequences originally identified as binding sites for the transcription factor MAZ both pause Pol II and activate polyadenylation of an upstream poly(A) signal. They do not affect polyadenylation efficiency in an uncoupled cleavage assay. In contrast, pausing of Pol II elongation induced by a high-affinity DNA-binding protein does not activate polyadenylation, indicating that G-rich MAZ sequences have a specific effect on polyadenylation. They also promote intrinsic pausing of purified Pol II, indicating a general role in the modulation of cotranscriptional RNA processing events.

Transcriptional termination signals for RNA polymerase II in fission yeast.

Transcription 'run-on' (TRO) analysis using permeabilized yeast cells indicates that transcription terminates between 180 and 380 bp downstream of the poly(A) site of the Schizosaccharomyces pombe ura4 gene. Two signals direct RNA polymerase II (pol II) to stop transcription: the previously identified 3' end formation signals located close to the poly(A) site and an additional downstream element (DSE) located at the region of termination. The downstream signal (135 bp) appears to act by pausing the elongating polymerase. TRO analysis indicates that elevated levels of transcribing polymerases accumulate over the DSE and that removal of this signal leads to transcription proceeding beyond the normal termination region. Furthermore, when inserted between two competing polyadenylation signals, this DSE increases the utilization of upstream poly(A) sites in vivo. We show that polymerase pausing over an extended region of template ensures termination of pol II transcription close to the poly(A) site.

Single-stranded RNA Recognition by the Bacteriophage T4 Translational Repressor, RegA

The T4 protein, RegA, is a translational repressor that blocks ribosome binding to multiple T4 messages by interacting with the mRNAs near their respective AUG start codons. Other than the AUG, there are no obvious similarities between the affected mRNAs. High affinity RNA ligands to RegA were isolated using SELEX (systematic evolution of ligands by exponential enrichment). The selected RNAs exhibited the consensus sequence 5'-AAAAUUGUUAUGUAA-3'. The AUG was invariant, suggesting that it is the primary effector of binding specificity. The UU immediately 5' to the AUG and the upstream poly(A) tract were highly conserved among the selected RNAs. Boundary and footprinting experiments are consistent with the consensus sequence defining the RegA-binding site. Interestingly, chemical modification and nuclease digestion data indicate that the RNA-binding site is single-stranded, as if RegA discriminates between targets based on their primary sequence, not their secondary structure. Minor variations from the consensus at positions other than the universally conserved AUG have little effect on RegA binding, but accumulation of mutations has a profound effect on the interaction. Comparison of the in vivo targets for RegA to the SELEX-generated consensus suggests a repression pattern whereby the translation of individual messages is sequentially halted until the least similarly affected message, the regA gene itself, is repressed.