A simple, robust, broadly applicable insertion mutagenesis method to create random fluorescent protein: target protein fusions.

A simple, broadly applicable method was developed using an in vitro transposition reaction followed by transformation into Escherichia coli and screening plates for fluorescent colonies. The transposition reaction catalyzes the random insertion of a fluorescent protein open reading frame into a target gene on a plasmid. The transposition reaction is employed directly in an E. coli transformation with no further procedures. Plating at high colony density yields fluorescent colonies. Plasmids purified from fluorescent colonies contain random, in-frame fusion proteins into the target gene. The plate screen also results in expressed, stable proteins. A large library of chimeric proteins was produced, which was useful for downstream research. The effect of using different fluorescent proteins was investigated as well as the dependence of the linker sequence between the target and fluorescent protein open reading frames. The utility and simplicity of the method were demonstrated by the fact that it has been employed in an undergraduate biology laboratory class without failure over dozens of class sections. This suggests that the method will be useful in high-impact research at small liberal arts colleges with limited resources. However, in-frame fusion proteins were obtained from 8 different targets suggesting that the method is broadly applicable in any research setting.

Mutations in yeast Pcf11, a conserved protein essential for mRNA 3' end processing and transcription termination, elicit the Environmental Stress Response.

The Pcf11 protein is an essential subunit of the large complex that cleaves and polyadenylates eukaryotic mRNA precursor. It has also been functionally linked to gene-looping, termination of RNA Polymerase II (Pol II) transcripts, and mRNA export. We have examined a poorly characterized but conserved domain (amino acids 142-225) of the Saccharomyces cerevisiae  Pcf11 and found that while it is not needed for mRNA 3' end processing or termination downstream of the poly(A) sites of protein-coding genes, its presence improves the interaction with Pol II and the use of transcription terminators near gene promoters. Analysis of genome-wide Pol II occupancy in cells with Pcf11 missing this region, as well as Pcf11 mutated in the Pol II CTD Interacting Domain, indicates that systematic changes in mRNA expression are mediated primarily at the level of transcription. Global expression analysis also shows that a general stress response, involving both activation and suppression of specific gene sets known to be regulated in response to a wide variety of stresses, is induced in the two pcf11 mutants, even though cells are grown in optimal conditions. The mutants also cause an unbalanced expression of cell wall-related genes that does not activate the Cell Wall Integrity pathway but is associated with strong caffeine sensitivity. Based on these findings, we propose that Pcf11 can modulate the expression level of specific functional groups of genes in ways that do not involve its well-characterized role in mRNA 3' end processing.

RNA polymerase II transcription attenuation at the yeast DNA repair gene DEF1 is biologically significant and dependent on the Hrp1 RNA-recognition motif.

Premature transcription termination (i.e. attenuation) is a potent gene regulatory mechanism that represses mRNA synthesis. Attenuation of RNA polymerase II is more prevalent than once appreciated, targeting 10-15% of mRNA genes in yeast through higher eukaryotes, but its significance and mechanism remain obscure. In the yeast Saccharomyces cerevisiae, polymerase II attenuation was initially shown to rely on Nrd1-Nab3-Sen1 termination, but more recently our laboratory characterized a hybrid termination pathway involving Hrp1, an RNA-binding protein in the 3'-end cleavage factor. One of the hybrid attenuation gene targets is DEF1, which encodes a repair protein that promotes degradation of polymerase II stalled at DNA lesions. In this study, we characterized the chromosomal DEF1 attenuator and the functional role of Hrp1. DEF1 attenuator mutants overexpressed Def1 mRNA and protein, exacerbated polymerase II degradation, and hindered cell growth, supporting a biologically significant DEF1 attenuator function. Using an auxin-induced Hrp1 depletion system, we identified new Hrp1-dependent attenuators in MNR2, SNG1, and RAD3 genes. An hrp1-5 mutant (L205S) known to impair binding to cleavage factor protein Rna14 also disrupted attenuation, but surprisingly no widespread defect was observed for an hrp1-1 mutant (K160E) located in the RNA-recognition motif. We designed a new RNA recognition motif mutant (hrp1-F162W) that altered a highly conserved residue and was lethal in single copy. In a heterozygous strain, hrp1-F162W exhibited dominant-negative readthrough defects at several gene attenuators. Overall, our results expand the hybrid RNA polymerase II termination pathway, confirming that Hrp1-dependent attenuation controls multiple yeast genes and may function through binding cleavage factor proteins and/or RNA.

RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination.

Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae, Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DEF1 DNA repair gene, which includes a portion of the 5'-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a DEF1 attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating DEF1 expression, which can be modulated during the DNA damage response.

Stimulation of RNA Polymerase II ubiquitination and degradation by yeast mRNA 3'-end processing factors is a conserved DNA damage response in eukaryotes.

The quality and retrieval of genetic information is imperative to the survival and reproduction of all living cells. Ultraviolet (UV) light induces lesions that obstruct DNA access during transcription, replication, and repair. Failure to remove UV-induced lesions can abrogate gene expression and cell division, resulting in permanent DNA mutations. To defend against UV damage, cells utilize transcription-coupled nucleotide excision repair (TC-NER) to quickly target lesions within active genes. In cases of long-term genotoxic stress, a slower alternative pathway promotes degradation of RNA Polymerase II (Pol II) to allow for global genomic nucleotide excision repair (GG-NER). The crosstalk between TC-NER and GG-NER pathways and the extent of their coordination with other nuclear events has remained elusive. We aimed to identify functional links between the DNA damage response (DDR) and the mRNA 3'-end processing complex. Our labs have previously shown that UV-induced inhibition of mRNA processing is a conserved DDR between yeast and mammalian cells. Here we have identified mutations in the yeast mRNA 3'-end processing cleavage factor IA (CFIA) and cleavage and polyadenylation factor (CPF) that confer sensitivity to UV-type DNA damage. In the absence of TC-NER, CFIA and CPF mutants show reduced UV tolerance and an increased frequency of UV-induced genomic mutations, consistent with a role for RNA processing factors in an alternative DNA repair pathway. CFIA and CPF mutants impaired the ubiquitination and degradation of Pol II following DNA damage, but the co-transcriptional recruitment of Pol II degradation factors Elc1 and Def1 was undiminished. Overall these data are consistent with yeast 3'-end processing factors contributing to the removal of Pol II stalled at UV-type DNA lesions, a functional interaction that is conserved between homologous factors in yeast and human cells.

DNA damage induces targeted, genome-wide variation of poly(A) sites in budding yeast.

Systemic response to DNA damage and other stresses is a complex process that includes changes in the regulation and activity of nearly all stages of gene expression. One gene regulatory mechanism used by eukaryotes is selection among alternative transcript isoforms that differ in polyadenylation [poly(A)] sites, resulting in changes either to the coding sequence or to portions of the 3' UTR that govern translation, stability, and localization. To determine the extent to which this means of regulation is used in response to DNA damage, we conducted a global analysis of poly(A) site usage in Saccharomyces cerevisiae after exposure to the UV mimetic, 4-nitroquinoline 1-oxide (4NQO). Two thousand thirty-one genes were found to have significant variation in poly(A) site distributions following 4NQO treatment, with a strong bias toward loss of short transcripts, including many with poly(A) sites located within the protein coding sequence (CDS). We further explored one possible mechanism that could contribute to the widespread differences in mRNA isoforms. The change in poly(A) site profile was associated with an inhibition of cleavage and polyadenylation in cell extract and a decrease in the levels of several key subunits in the mRNA 3'-end processing complex. Sequence analysis identified differences in the cis-acting elements that flank putatively suppressed and enhanced poly(A) sites, suggesting a mechanism that could discriminate between variable and constitutive poly(A) sites. Our analysis indicates that variation in mRNA length is an important part of the regulatory response to DNA damage.

Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology.

Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. On the basis of our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1.

Unravelling the means to an end: RNA polymerase II transcription termination.

The pervasiveness of RNA synthesis in eukaryotes is largely the result of RNA polymerase II (Pol II)-mediated transcription, and termination of its activity is necessary to partition the genome and maintain the proper expression of neighbouring genes. Despite its ever-increasing biological significance, transcription termination remains one of the least understood processes in gene expression. However, recent mechanistic studies have revealed a striking convergence among several overlapping models of termination, including the poly(A)- and Sen1-dependent pathways, as well as new insights into the specificity of Pol II termination among its diverse gene targets. Broader knowledge of the role of Pol II carboxy-terminal domain phosphorylation in promoting alternative mechanisms of termination has also been gained.

Money, sex, and drugs: a case study to teach the genetics of antibiotic resistance.

The goal of the work reported here was to help students expand their understanding of antibiotic resistance, the Central Dogma, and evolution. We developed a unit entitled "Ciprofloxacin Resistance in Neisseria gonorrhoeae," which was constructed according to the principles of scientific teaching by a team of graduate students, science faculty, and instructors. A variety of activities and assessments were used, including a case study, short lectures, and group problem-solving. Implementation of "Ciprofloxacin Resistance in Neisseria gonorrhoeae" in a college freshman seminar suggests these materials are useful in increasing understanding of complex biological topics and improving problem-solving abilities.

Regulation of a eukaryotic gene by GTP-dependent start site selection and transcription attenuation.

Guanine nucleotide negatively regulates yeast inosine monophosphate dehydrogenase (IMPDH) mRNA synthesis by an unknown mechanism. IMPDH catalyzes the first dedicated step of GTP biosynthesis, and feedback control of its expression maintains the proper balance of purine nucleotides. Here we show that RNA polymerase II (Pol II) responds to GTP concentration. When GTP is sufficient, Pol II initiates transcription of the IMPDH gene (IMD2) at TATA box-proximal "G" sites, producing attenuated transcripts. When GTP is deficient, Pol II initiates at an "A" further downstream, circumventing the regulatory terminator to produce IMPDH mRNA. A major determinant for GTP concentration-dependent initiation at the upstream sites is the presence of guanine at the first and second positions of the transcript. Mutations in the Rpb1 subunit of Pol II and in TFIIB disrupt IMD2 regulation by altering start site selection. Thus, Pol II initiation can be regulated by the concentration of initiating nucleotide.

Genome-wide distribution of yeast RNA polymerase II and its control by Sen1 helicase.

Functional engagement of RNA polymerase II (Pol II) with eukaryotic chromosomes is a fundamental and highly regulated biological process. Here we present a high-resolution map of Pol II occupancy across the entire yeast genome. We compared a wild-type strain with a strain bearing a substitution in the Sen1 helicase, which is a Pol II termination factor for noncoding RNA genes. The wild-type pattern of Pol II distribution provides unexpected insights into the mechanisms by which genes are repressed or silenced. Remarkably, a single amino acid substitution that compromises Sen1 function causes profound changes in Pol II distribution over both noncoding and protein-coding genes, establishing an important function of Sen1 in the regulation of transcription. Given the strong similarity of the yeast and human Sen1 proteins, our results suggest that progressive neurological disorders caused by substitutions in the human Sen1 homolog Senataxin may be due to misregulation of transcription.

Quantitative analysis of in vivo initiator selection by yeast RNA polymerase II supports a scanning model.

Initiation of transcription by RNA polymerase II (RNAP II) on Saccharomyces cerevisiae messenger RNA (mRNA) genes typically occurs at multiple sites 40-120 bp downstream of the TATA box. The mechanism that accommodates this extended and variable promoter architecture is unknown, but one model suggests that RNAP II forms an open promoter complex near the TATA box and then scans the template DNA strand for start sites. Unlike most protein-coding genes, small nuclear RNA gene transcription starts predominantly at a single position. We identify a highly efficient initiator element as the primary start site determinant for the yeast U4 small nuclear RNA gene, SNR14. Consistent with the scanning model, transcription of an SNR14 allele with tandemly duplicated start sites initiates primarily from the upstream site, yet the downstream site is recognized with equivalent efficiency by the diminished population of RNAP II molecules that encounter it. A quantitative in vivo assay revealed that SNR14 initiator efficiency is nearly perfect (approximately 90%), which explains the precision of U4 RNA 5' end formation. Initiator efficiency was reduced by cis-acting mutations at -8, -7, -1, and +1 and trans-acting substitutions in the TFIIB B-finger. These results expand our understanding of RNAP II initiation preferences and provide new support for the scanning model.