The cell: an image library-CCDB: a curated repository of microscopy data.

The cell: an image library-CCDB (CIL-CCDB) (http://www.cellimagelibrary.org) is a searchable database and archive of cellular images. As a repository for microscopy data, it accepts all forms of cell imaging from light and electron microscopy, including multi-dimensional images, Z- and time stacks in a broad variety of raw-data formats, as well as movies and animations. The software design of CIL-CCDB was intentionally designed to allow easy incorporation of new technologies and image formats as they are developed. Currently, CIL-CCDB contains over 9250 images from 358 different species. Images are evaluated for quality and annotated with terms from 14 different ontologies in 16 different fields as well as a basic description and technical details. Since its public launch on 9 August 2010, it has been designed to serve as not only an archive but also an active site for researchers and educators.

50 Years of Women in Cell Biology: Where have we been? Where are we going?

It's been 50 years since Women in Cell Biology (WICB) was founded by junior women cell biologists who found themselves neither represented at the American Society for Cell Biology (ASCB) presentations nor receiving the information, mentoring, and sponsorship they needed to advance their careers. Since then, gender parity at ASCB has made significant strides: WICB has become a standing ASCB committee, women are regularly elected president of the ASCB, and half the symposia speakers are women. Many of WICB's pioneering initiatives for professional development, including career panels, workshops, awards for accomplishments in science and mentoring, and career mentoring roundtables, have been incorporated and adapted into broader "professional development" that benefits all members of ASCB. The time has passed when we can assume that all women benefit equally from progress. By strategically, thoughtfully, and honestly recognizing the challenges to women of the past and today, we may anticipate those new challenges that will arise in the next 50 years. WICB, in collaboration with the ASCB, can lead in data collection and access and can promote diversity, equity, and inclusion. This work will be a fitting homage to the women who, half a century ago, posted bathroom stall invitations to the first Women in Cell Biology meetup.

The YEATS domain of Taf14 in Saccharomyces cerevisiae has a negative impact on cell growth.

The role of a highly conserved YEATS protein motif is explored in the context of the Taf14 protein of Saccharomyces cerevisiae. In S. cerevisiae, Taf14 is a protein physically associated with many critical multisubunit complexes including the general transcription factors TFIID and TFIIF, the chromatin remodeling complexes SWI/SNF, Ino80 and RSC, Mediator and the histone modification enzyme NuA3. Taf14 is a member of the YEATS superfamily, conserved from bacteria to eukaryotes and thought to have a transcription stimulatory activity. However, besides its ubiquitous presence and its links with transcription, little is known about Taf14's role in the nucleus. We use structure-function and mutational analysis to study the function of Taf14 and its well conserved N-terminal YEATS domain. We show here that the YEATS domain is not necessary for Taf14's association with these transcription and chromatin remodeling complexes, and that its presence in these complexes is dependent only on its C-terminal domain. Our results also indicate that Taf14's YEATS domain is not necessary for complementing the synthetic lethality between TAF14 and the general transcription factor TFIIS (encoded by DST1). Furthermore, we present evidence that the YEATS domain of Taf14 has a negative impact on cell growth: its absence enables cells to grow better than wild-type cells under stress conditions, like the microtubule destabilizing drug benomyl. Moreover, cells expressing solely the YEATS domain grow worser than cells expressing any other Taf14 construct tested, including the deletion mutant. Thus, this highly conserved domain should be considered part of a negative regulatory loop in cell growth.

Running with RNA polymerase: eukaryotic transcript elongation.

Long recognized as a target of regulation in prokaryotes, transcript elongation has recently become the focus of many investigators interested in eukaryotic gene expression. The growth of this area has been fueled by the availability of new methods and molecular structures, expanding sequence databases and an appreciation for the exquisite coordination required among different processes in the nucleus. Our article collates new information on regulatory accessory factors, as well as their ultimate target, RNA polymerase, in the nucleus of eukaryotic cells. How this regulation influences the biology of the organism is quite profound, and from single cell to multicellular eukaryotes significant similarities exist in the molecular responses to extracellular signals during transcript elongation. The most advanced genetic knowledge in this area comes from Saccharomyces cerevisiae, but the biochemistry and cell biology results from other organisms are also highlighted.

Genetic interactions between TFIIF and TFIIS.

The eukaryotic transcript elongation factor TFIIS is encoded by a nonessential gene, PPR2, in Saccharomyces cerevisiae. Disruptions of PPR2 are lethal in conjunction with a disruption in the nonessential gene TAF14/TFG3. While investigating which of the Taf14p-containing complexes may be responsible for the synthetic lethality between ppr2Delta and taf14Delta, we discovered genetic interactions between PPR2 and both TFG1 and TFG2 encoding the two larger subunits of the TFIIF complex that also contains Taf14p. Mutant alleles of tfg1 or tfg2 that render cells cold sensitive have improved growth at low temperature in the absence of TFIIS. Remarkably, the amino-terminal 130 amino acids of TFIIS, which are dispensable for the known in vitro and in vivo activities of TFIIS, are required to complement the lethality in taf14Delta ppr2Delta cells. Analyses of deletion and chimeric gene constructs of PPR2 implicate contributions by different regions of this N-terminal domain. No strong common phenotypes were identified for the ppr2Delta and taf14Delta strains, implying that the proteins are not functionally redundant. Instead, the absence of Taf14p in the cell appears to create a dependence on an undefined function of TFIIS mediated by its N-terminal region. This region of TFIIS is also at least in part responsible for the deleterious effect of TFIIS on tfg1 or tfg2 cold-sensitive cells. Together, these results suggest a physiologically relevant functional connection between TFIIS and TFIIF.

Promoting elongation with transcript cleavage stimulatory factors.

Transcript elongation by RNA polymerase is a dynamic process, capable of responding to a number of intrinsic and extrinsic signals. A number of elongation factors have been identified that enhance the rate or efficiency of transcription. One such class of factors facilitates RNA polymerase transcription through blocks to elongation by stimulating the polymerase to cleave the nascent RNA transcript within the elongation complex. These cleavage factors are represented by the Gre factors from prokaryotes, and TFIIS and TFIIS-like factors found in archaea and eukaryotes. High-resolution structures of RNA polymerases and the cleavage factors in conjunction with biochemical investigations and genetic analyses have provided insights into the mechanism of action of these elongation factors. However, there are yet many unanswered questions regarding the regulation of these factors and their effects on target genes.

Evaluating a science diversity program at UC Berkeley: more questions than answers.

For the past three decades, much attention has been focused on developing diversity programs designed to improve the academic success of underrepresented minorities, primarily in mathematics, science, and engineering. However, ethnic minorities remain underrepresented in science majors and careers. Over the last 10 years, the Biology Scholars Program (BSP), a diversity program at the University of California (UC), Berkeley, has worked to increase the participation and success of students majoring in the biological sciences. A quantitative comparison of students in and out of the program indicates that students in BSP graduate with a degree in biology at significantly higher rates than students not in BSP regardless of race/ethnicity. Furthermore, students who are in BSP have statistically lower high school grade point averages (GPAs) and Scholastic Achievement Test (SAT) scores than students not in BSP. African-American and Hispanic students who join BSP graduate with significantly higher UC Berkeley biology GPAs than non-BSP African-American and Hispanic students, respectively. Majority (Asian and White) students in BSP graduate with statistically similar UC GPAs despite having lower SAT scores than non-BSP majority students. Although BSP students are more successful in completing a biology degree than non-program members, the results raise a series of questions about why the program works and for whom.

The Stimulatory Mechanism of Hepatitis C Virus NS5A Protein on the NS5B Catalyzed Replication Reaction In Vitro.

The Hepatitis C Virus RNA dependent RNA polymerase, NS5B, is stimulated by the NS5A protein in vitro. To explore this stimulatory mechanism, we compared the activity of a mutant of NS5B containing a deletion of the ß-loop region with that of the full length NS5B in response to NS5A. While the NS5A protein does stimulate full length NS5B, NS5A does not stimulate the NS5B deletion mutant during either replication initiation or elongation. This result suggests that the activation mechanism might involve a NS5A-mediated conformational change of the ß-loop of NS5B. Such a conformational change would be predicted to prevent steric clash of the RNA template and newly synthesized RNA product. Consistent with this hypothesis, RNA binding is enhanced when the full length NS5B and NS5A are incubated with RNA, but RNA binding is unchanged with incubation of NS5A and the NS5B ß-loop deletion mutant.

The Hepatitis C Virus NS5A Stimulates NS5B During In Vitro RNA Synthesis in a Template Specific Manner.

The hepatitis C virus (HCV) NS5B protein contains the RNA dependent RNA polymerase (RdRp) activity that catalyzes the synthesis of the viral genome with other host and viral factors. NS5A is an HCV-encoded protein previously shown to localize to the replisome and be necessary for viral replication. However, its role in replication has not been defined. Using an in vitro biochemical assay, we detected a stimulatory effect of NS5A on the NS5B replication reaction with minimal natural templates. NS5A stimulates replication by NS5B on two templates derived from the 3' end of the RNA genome (4 fold +/- 1.3 fold). A pre-incubation step with the two proteins prior to the replication reaction and substoichiometric levels of NS5A are required for detecting stimulation. With a template derived from the 3'end complementary to the RNA genome (the negative strand) no stimulation was observed. Furthermore, with a synthetic template that allows studying different phases of replication, NS5A stimulates NS5B during elongation. These findings suggest that NS5A stimulates NS5B during synthesis of the complementary (i.e., negative) strand of the RNA genome.

Transcript elongation factor TFIIS is involved in arabidopsis seed dormancy.

Transcript elongation factor TFIIS promotes efficient transcription by RNA polymerase II, since it assists in bypassing blocks during mRNA synthesis. While yeast cells lacking TFIIS are viable, inactivation of mouse TFIIS causes embryonic lethality. Here, we have identified a protein encoded in the Arabidopsis genome that displays a marked sequence similarity to TFIIS of other organisms, primarily within domains II and III in the C-terminal part of the protein. TFIIS is widely expressed in Arabidopsis, and a green fluorescent protein-TFIIS fusion protein localises specifically to the cell nucleus. When expressed in yeast cells lacking the endogenous TFIIS, Arabidopsis TFIIS partially complements the sensitivity of mutant cells to the nucleotide analog 6-azauridine, which is a typical characteristic of transcript elongation factors. We have characterised Arabidopsis lines harbouring T-DNA insertions in the coding sequence of TFIIS. Plants homozygous for T-DNA insertions are viable, and genomewide transcript profiling revealed that compared to control plants, a relatively small number of genes are differentially expressed in mutant plants. TFIIS(-/-) plants display essentially normal development, but they flower slightly earlier than control plants and show clearly reduced seed dormancy. Plants with RNAi-mediated knockdown of TFIIS expression also are affected in seed dormancy. Therefore, TFIIS plays a critical role in Arabidopsis seed development.

Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae.

Unknown mechanisms exist to ensure that exons are not skipped during biogenesis of mRNA. Studies have connected transcription elongation with regulated alternative exon inclusion. To determine whether the relative rates of transcription elongation and spliceosome assembly might play a general role in enforcing constitutive exon inclusion, we measured exon skipping for a natural two-intron gene in which the internal exon is constitutively included in the mRNA. Mutations in this gene that subtly reduce recognition of the intron 1 branchpoint cause exon skipping, indicating that rapid recognition of the first intron is important for enforcing exon inclusion. To test the role of transcription elongation, we treated cells to increase or decrease the rate of transcription elongation. Consistent with the "first come, first served" model, we found that exon skipping in vivo is inhibited when transcription is slowed by RNAP II mutants or when cells are treated with inhibitors of elongation. Expression of the elongation factor TFIIS stimulates exon skipping, and this effect is eliminated when lac repressor is targeted to DNA encoding the second intron. A mutation in U2 snRNA promotes exon skipping, presumably because a delay in recognition of the first intron allows elongating RNA polymerase to transcribe the downstream intron. This indicates that the relative rates of elongation and splicing are tuned so that the fidelity of exon inclusion is enhanced. These findings support a general role for kinetic coordination of transcription elongation and splicing during the transcription-dependent control of splicing.

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 1. RNA chain initiation, abortive initiation, and promoter escape at three bacteriophage promoters.

RNA chain initiation and promoter escape is the latter stage of transcription initiation. This stage is characterized by several well-defined biochemical events: synthesis and release of short RNA products ranging 2 to 15 nucleotides in length, release of the sigma subunit from the enzyme-promoter complex, and initial translocation of the polymerase away from the promoter. In this paper, we report the use of a steady-state transcription assay with [gamma-(32)P]ATP labeling to subject the RNA chain initiation-promoter escape reaction to quantitative analysis. The specific parameters we follow to describe the chain initiation-promoter escape process include the abortive and productive rates, the abortive probability, the abortive:productive ratio, and the maximal size of the abortive product. In this study, we measure these parameters for three bacteriophage promoters transcribed by Escherichia coli RNA polymerase: T7 A1, T5 N25, and T5 N25(antiDSR). Our studies show that all three promoters form substantial amounts of abortive products under all conditions we tested. However, each of the promoters shows distinct differences from the others when the various parameters are compared. At 100 microM NTP, in a 10 min reaction, the abortive and productive yields are 87 and 13%, respectively, for T7 A1; 97 and 3%, respectively, for T5 N25; and 99.4 and 0.6%, respectively, for T5 N25(antiDSR). These values correspond to approximately 7, 32, and 165 abortive transcripts per productive transcript for the three promoters, respectively. The yield of most of the abortive products is not affected by the elevated concentration of the NTP substrate corresponding to the next template-specified nucleotide; hence, abortive products are not normally formed through a simple process of "kinetic competition". Instead, formation of abortive products appears to be determined by intrinsic DNA signals embedded in the promoter recognition region and the initial transcribed sequence region of each promoter.

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 2. Formation and characterization of two distinct classes of initial transcribing complexes.

By following the kinetics of abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term "productive ITC" and "unproductive ITC". The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of abortive initiation steps. The unproductive ITCs were found to synthesize mostly abortive transcripts of 2-3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproductive ITCs most likely during the formation of open promoter complexes. The extent of partitioning varies with individual promoter sequences and is dependent on the nature and concentration of the initiating nucleotide. Thus, multiple classes of ITCs can be formed during promoter binding and transcript initiation.

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 3. Influences of individual DNA elements within the promoter recognition region on abortive initiation and promoter escape.

Abortive initiation and promoter escape are two principal biochemical reactions occurring in the latter stage of transcript initiation. We have analyzed the influences of individual DNA elements within the promoter recognition region (PRR) on these reactions by measuring the quantitative initiation parameters that describe abortive initiation and promoter escape; these parameters are the abortive rate, the productive rate, the abortive:productive ratio, the abortive probability, and the maximum size of abortive transcripts. Changes in the individual DNA elements within the PRR can have a substantial effect on each of these parameters. The discriminator region and the -10 element primarily influence the abortive probability at positions 2-5 and 6-10, respectively, while the -10 and -35 conserved hexamers and the spacer region affect the abortive probability at positions 11-15. Surprisingly, transcription of a consensus promoter invariably gives a higher abortive yield, a higher abortive probability, a longer abortive ladder, and a lower productive rate than promoter variants carrying even a single deviation in the consensus hexamers. These results suggest that strong RNA polymerase-PRR interactions stall the polymerase at the promoter, thereby reducing the rate of promoter escape and consequently enhancing the extent of abortive initiation.

Intrinsic transcript cleavage in yeast RNA polymerase II elongation complexes.

Transcript elongation can be interrupted by a variety of obstacles, including certain DNA sequences, DNA-binding proteins, chromatin, and DNA lesions. Bypass of many of these impediments is facilitated by elongation factor TFIIS through a mechanism that involves cleavage of the nascent transcript by the RNA polymerase II/TFIIS elongation complex. Highly purified yeast RNA polymerase II is able to perform transcript hydrolysis in the absence of TFIIS. The "intrinsic" cleavage activity is greatly stimulated at mildly basic pH and requires divalent cations. Both arrested and stalled complexes can carry out the intrinsic cleavage reaction, although not all stalled complexes are equally efficient at this reaction. Arrested complexes in which the nascent transcript was cleaved in the absence of TFIIS were reactivated to readthrough blocks to elongation. Thus, cleavage of the nascent transcript is sufficient for reactivating some arrested complexes. Small RNA products released following transcript cleavage in stalled ternary complexes differ depending upon whether the cleavage has been induced by TFIIS or has occurred in mildly alkaline conditions. In contrast, both intrinsic and TFIIS-induced small RNA cleavage products are very similar when produced from an arrested ternary complex. Although alpha-amanitin interferes with the transcript cleavage stimulated by TFIIS, it has little effect on the intrinsic cleavage reaction. A mutant RNA polymerase previously shown to be refractory to TFIIS-induced transcript cleavage is essentially identical to the wild type polymerase in all tested aspects of intrinsic cleavage.