Thomas Linn

A set of compatible tac promoter expression vectors.

A series of expression vectors have been developed which all contain an identical expression cassette comprised of the lacIq gene, the tac promoter, a multiple cloning site (MCS) and a downstream transcriptional terminator. This cassette has been inserted into four distinct plasmid backbones, each of which is from a separate incompatibility group and carries a different drug resistance gene. Therefore, different combinations of these expression plasmids can be stably maintained together.

ANOVA-like differential expression (ALDEx) analysis for mixed population RNA-Seq.

Experimental variance is a major challenge when dealing with high-throughput sequencing data. This variance has several sources: sampling replication, technical replication, variability within biological conditions, and variability between biological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observed variance into these categories as per standard ANOVA models. We show that the partitioning of within-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments, and further find that commonly-used RNA-Seq analysis tools, as described in the literature, do not enforce the constraint that the sum of relative expression levels must be one, and thus report expression levels that are systematically distorted. These two factors lead to misleading inferences if not properly accommodated. As it is usually only the biological between-condition and within-condition differences that are of interest, we developed ALDEx, an ANOVA-like differential expression procedure, to identify genes with greater between- to within-condition differences. We show that the presence of differential expression and the magnitude of these comparative differences can be reasonably estimated with even very small sample sizes.

Synthesis of the β and β′ subunits of Escherichia coli RNA polymerase is autogenously regulated in vivo by both transcriptional and translational mechanisms

Numerous experiments have indicated that the synthesis of RNA polymerase (ββ′α2σ70) in Escherichia coli is autogenously regulated. In the present study, we have examined expression of the rpoB and rpoC genes which encode the β and β′ subunits of RNA polymerase. These genes are the distal cistrons of the rplKAJLrpoBC ribosomal protein–RNA polymerase transcription unit. Both transcriptional (operon) and translational (gene) fusions of either rpoB or rpoC to the lacZ reporter were used to monitor their in vivo expression by inserting single copies of these fusions into the bacterial chromosome on integration‐proficient lambda vectors. In order to examine the expression of the rpoBC genes in response to varied intracellular concentrations of the RNA polymerase subunits, a series of expression plasmids carrying the rpoBrpoCrpoA (α) and rpoD (σ70) genes in different combinations were then introduced into these cells. Elevated concentrations of either β or β′ were found to repress the expression of both rpoB and rpoC at the translational level. However, the simultaneous increase in the concentration of all the subunits that comprise holoenzyme repressed the transcription of rpoBC. To determine the site of this repression, additional operon fusions were utilized which placed lacZ downstream of each of the transcriptional regulatory sites of this gene cluster, including two promoters, rplKp and rplJp, and a transcriptional attenuator in the rplL–rpoB intercistronic region. Expression from these fusions showed that transcriptional repression is achieved primarily by reducing initiation at both upstream promoters, coupled with a small increase in termination at the attenuator.

Growth rate-dependent regulation of RNA polymerase synthesis in Escherichia coli

SummaryThe rate of synthesis of the β and β′ subunits of RNA polymerase relative to the rate of synthesis of total protein was found to remain constant with increasing steady state growth rate. This is in contrast to the relative synthesis rates of ribosomal proteins which are known to increase with growth rate. Yet the ratio of the rate of transcription of the ribosomal protein (rplJL) and RNA polymerase (rpoBC) domains of the rplKAJLrpoBC gene cluster was found to be invariant. Fusions to lacZ were used to relate the rate of transcription of the rplKAJL genes to the rate of synthesis of total protein. No change was seen at growth rates above 0.8 doublings per hour. This indicates that the growth rate-dependent expression of these ribosomal proteins is regulated at the post-transcriptional level. However because both the relative rate of transcription of rpoBC and rate of synthesis of β and β′ were found to remain invariant over this growth range it suggests the expression of these RNA polymerase subunits is regulated at the transcriptional level.

A refined vector system for the in vitro construction of single-copy transcriptional or translational fusions to lacZ

New single-copy vectors based on lambda phage have been developed for creating either transcriptional (operon) or translational (gene) fusions to the lacZ gene. The improvements of these vectors over the previous lambda TL61 vector include: (i) incorporation of a tetracycline-resistance-encoding gene (TcR) to permit direct selection of lysogens, (ii) low-background beta-galactosidase activity, (iii) the ability to accept DNA inserts up to 8 kb in size, and (iv) an expanded multiple cloning site (MCS). The new transcriptional fusion vector retains the RNase III processing site downstream from the MCS which ensures independent translation of lacZ. The set of three translational fusion vectors allow for convenient subcloning in any of the three translational reading frames.

In vivo analysis of overlapping transcription units in the rplKAJLrpoBC ribosomal protein-RNA polymerase gene cluster of Escherichia coli.

Transcription of the rplKAJLrpoBC ribosomal protein (rpl) RNA polymerase (rpo) gene cluster is governed by a complex set of signals. To dissect the transcription units active in vivo and to quantify the relative contribution of each, an extensive array of rplKAJLrpoB/lacZ gene fusions were constructed on lambda phage derivatives and introduced in single copy into the chromosomes of lac- cells. Measurements of beta-galactosidase production from fusions containing wild-type and/or mutagenized rplrpo DNA fragments permitted the establishment of high-resolution transcription profiles of the gene cluster. The results show that transcription initiated at the upstream rplKp promoter (located just before rplK) does not terminate before the rplJp promoter (located upstream from rplJ), but instead reads through into the distal genes. In addition, rplJp continues to function efficiently in the presence of readthrough transcription from rplKp. As a result the rplJL genes are transcribed at almost twice the frequency of the upstream rplKA genes. However, the transcription of rpoB, which is situated downstream from the previously identified attenuator (rpoBa), is only marginally increased (20%) when both promoters are present. This suggests that although both transcription units overlap, transcriptional termination at rpoBa is modulated in response to the frequency of initiation from both promoters.

Transcription-frequency-dependent modulation of an attenuator in a ribosomal protein-RNA polymerase operon requires an upstream site

Although the attenuator located between the ribosomal protein and RNA polymerase gene domains of the Escherichia coli rplKAJLrpoBC operon has a maximum termination efficiency of 80%, the level of termination is diminished with decreasing transcription frequency. In this report, the use of transcriptional fusions to further investigate the mechanism of transcription-frequency-dependent regulation is described. The termination efficiency of two other weak terminators was assayed over a wide range of transcription frequencies programmed by different strength promoters. The results indicated that a decrease in termination efficiency with decreasing transcription frequency is not an inherent property of weak terminators. Deletion of the 165 bp segment located 439-274 bp upstream of the attenuator abrogated the difference in termination efficiency normally seen between high and low levels of transcription. This suggests that a cis-acting site located in this upstream region is necessary for transcription-frequency-dependent modulation of the attenuators function. However, this site apparently works only in combination with the attenuator, since it did not cause transcription-frequency-dependent modulation when placed upstream of two other weak terminators. Analysis of the readthrough frequencies of single or tandem copies of the attenuator indicated that the transcription complexes which pass through the attenuator have not been converted to termination-resistant complexes in a manner analogous to the N-mediated antitermination system of lambda. Finally, an examination of termination efficiency in three nusA mutants suggested that although NusA increases readthrough at the attenuator it is not directly involved in transcription-frequency-dependent modulation.