RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing.

Previous investigations into the mechanisms that control RNA Polymerase (Pol) I transcription have primarily focused on the process of transcription initiation, thus little is known regarding postinitiation steps in the transcription cycle. Spt4p and Spt5p are conserved throughout eukaryotes, and they affect elongation by Pol II. We have found that these two proteins copurify with Pol I and associate with the rDNA in vivo. Disruption of the gene for Spt4p resulted in a modest decrease in growth and rRNA synthesis rates at the permissive temperature, 30 degrees C. Furthermore, biochemical and EM analyses showed clear defects in rRNA processing. These data suggest that Spt4p, Spt5p, and, potentially, other regulators of Pol I transcription elongation play important roles in coupling rRNA transcription to its processing and ribosome assembly.

EM visualization of transcription by RNA polymerase II: downstream termination requires a poly(A) signal but not transcript cleavage.

We have used EM visualization of active genes on plasmid vectors in Xenopus oocyte nuclei to investigate the relationship between poly(A) signals and RNA polymerase II transcription termination. Although a functional poly(A) signal is required for efficient termination, cotranscriptional RNA cleavage at the poly(A) site is not. Furthermore, the phenomena of termination and cotranscriptional RNA cleavage can be uncoupled, and the efficiency of both varies independently on different copies of the same plasmid template in the same oocyte nucleus. The combined observations are consistent with a scenario in which there is template-specific addition to Pol II (presumably at the promoter) of elongation and/or RNA processing factors, which are altered upon passage through a poly(A) signal, resulting in termination and, in some cases, cotranscriptional RNA cleavage.

EM visualization of transcriptionally active genes after injection into Xenopus oocyte nuclei.

This article has described methods in use in our lab for microinjection of genes into Xenopus oocyte nuclei followed by EM visualization of those genes by the Miller chromatin spreading method. We consider our efforts to be still developing, as we attempt to maximize the visualization of specific, active, mappable genes. One of our main goals at this time is to find a DNA sequence element that will ensure efficient Pol II termination so that the common problem of read-through transcription (as seen in Fig. 6) can be overcome. We currently are testing three different elements reported to have roles in transcription termination. The method is evolving as a unique and valuable approach to study gene expression and RNA processing at the level of individual genes and individual transcripts. Given the ability to manipulate both cis- and trans-acting factors prior to EM visualization, its potential is limited only by the somewhat labor-intensive nature of the method.

Metazoan rDNA enhancer acts by making more genes transcriptionally active.

Enhancers could, in principle, function by increasing the rate of reinitiation on individual adjacent active promoters or by increasing the probability that an adjacent promoter is activated for transcription. We have addressed this issue for the repetitive metazoan rDNA enhancer by microinjecting Xenopus oocytes with enhancer-less and enhancer-bearing genes and determining by EM the frequency that each gene type forms active transcription units and their transcript density. We use conditions where transcription requires the normal rDNA promoter and is stimulated 30-50-fold by the enhancer. (In contrast, at saturating template conditions as used in previous EM studies, an aberrant mode of transcription is activated that is not affected by the rDNA enhancer or by the generally recognized rDNA promoter). The active transcription units on enhancer-less genes are found to be as densely packed with nascent transcripts and polymerases as those on enhancer-bearing genes and on the endogenous rRNA genes. Significantly, the enhancer-bearing genes are approximately 30-50-fold more likely to form such active transcription units than enhancer-less genes, consistent with their amounts of transcript. Complementary studies confirm that the enhancer does not affect elongation rate, the stability of the transcription complex, or transcript half-life. These data demonstrate that the repetitive metazoan rDNA enhancer causes more genes to be actively transcribed and does not alter the reinitiation rate on individual active genes.

Chromosome structure: DNA nucleotide sequence elements of a subset of the minichromosomes of the protozoan Trypanosoma brucei.

The genome of the protozoan Trypanosoma brucei contains a set of about 100 minichromosomes of about 50 to 150 kb in size. The small size of these chromosomes, their involvement in antigenic variation, and their mitotic stability make them ideal candidates for a structural analysis of protozoan chromosomes and their telomeres. We show that a subset of the minichromosomes is composed predominantly of simple-sequence DNA, with over 90% of the length of the minichromosome consisting of a tandem array of 177-bp repeats, indicating that these molecules have limited protein-coding capacity. Proceeding from the tip of the telomere to a chromosome internal position, a subset of the minichromosomes contained the GGGTTA telomere repeat, a 29-bp telomere-derived repeat, a region containing 74-bp G + C-rich direct repeats separated by approximately 155 bp of A + T-rich DNA that has a bent character, and 50 to 150 kb of the 177-bp repeat. Several of the minichromosome-derived telomeres did not encode protein-coding genes, indicating that the repertoire of telomeric variant cell surface glycoprotein genes is restricted to some telomeres only. The telomere organization in trypanosomes shares striking similarities to the organization of telomeres and subtelomeres in humans, yeasts, and plasmodia. An electron microscopic analysis of the minichromosomes showed that they are linear molecules without abnormal structures in the main body of the chromosome. The structure of replicating molecules indicated that minichromosomes probably have a single bidirectional origin of replication located in the body of the chromosome. We propose a model for the structure of the trypanosome minichromosomes.

Visualization of RNA transcription and processing.

Electron microscopic visualization of transcriptionally-active chromatin dispersed by the Miller spreading technique allows a unique view of in vivo genetic events on an individual gene basis. We have used the method to ultrastructurally analyze transcription, ribonucleoprotein assembly and early RNA processing events on the pre-messenger RNA transcripts of Drosophila melanogaster Pol II genes. Our findings are surprising in two regards--splicing as a rule initials co-transcriptionally and is frequently complete before polyadenylation, and cleavage at poly(A) sites, at least for a few specific genes, occurs post-transcriptionally.

EM analysis of Drosophila chorion genes: amplification, transcription termination and RNA splicing.

We have used the electron microscope to examine ultrastructurally several events occurring during the biogenesis of two very abundant chorion (eggshell) mRNA molecules in the follicle cells of Drosophila melanogaster--namely, selective gene amplification, transcription initiation and termination, and RNA processing. We find that the highly transcribed s36 and s38 genes are positioned in the central region of large, multi-forked amplified DNA structures. Transcript morphology is consistent with the known presence of a small intron at the 5' end of each gene. Mature transcripts are associated with spliceosomes, demonstrating that splice site selection occurs co-transcriptionally but that splicing is completed after transcript release from the template. We have also mapped the termination sites for the genes. The two genes exhibit efficient termination very near their poly(A) sites--within a 210 bp region for s36 and a 360 bp region for s38.

Visualization of Drosophila melanogaster chorion genes undergoing amplification.

We visualized by electron microscopy the preferential amplification of Drosophila chorion genes in late-stage follicle cells. Chromatin spreads revealed large clusters of actively transcribed genes of the appropriate size, spacing, and orientation for chorion genes that were expressed with the correct temporal specificity. Occasionally the active genes were observed within or contiguous with intact replicons and replication forks. In every case, our micrographs are consistent with the hypothesis that the central region of each chorion domain contains a replication origin(s) used during the amplification event. In one case, a small replication bubble was observed precisely at the site of the essential region of the X chromosome amplification control element. The micrographs also suggest that forks at either end of a replicon frequently progress very different distances, presumably due to different times in initiation or different rates of movement. It appears that all chorion genes (even those coding for minor proteins) are transcribed in a "fully on" condition, albeit for varied durations, and that if replication fork passage does inactivate a promoter, it does so very transiently. Furthermore, a DNA segment containing one active gene is likely to have an additional active gene(s). Surprisingly, during the time frame of expected maximum activity, approximately half of the chorion sequences appear transcriptionally inactive.

Splice site selection, rate of splicing, and alternative splicing on nascent transcripts.

Based on ultrastructural analysis of actively transcribing genes seen in electron micrographs, we present evidence that pre-mRNA splicing occurs with a reasonable frequency on the nascent transcripts of early Drosophila embryo genes and that splice site selection may generally precede polyadenylation. The details of the process observed are in agreement with results from in vitro splicing systems but differ in the more rapid completion of in vivo splicing. For those introns that are removed cotranscriptionally, a series of events is initiated following 3' splice site synthesis, beginning with ribonucleoprotein (RNP) particle formation at the 3' splice site within 48 sec, intron loop formation within 2 min, and splicing within 3 min. The initiation of the process is correlated with 3' splice site synthesis but is independent of 5' splice site synthesis, the position of the intron within the transcript, and the age or length of the transcript. In some cases, introns are removed from the 5' end of a transcript before introns are synthesized at the 3' end, supporting a possible role for the order of transcription in splice site pairing. In general, our observations are consistent with the 'first-come-first-served' principle of splice site selection, although an observed example of exon skipping indicates that alternative splicing possibilities can be accommodated within this general framework.

Two Drosophila chorion genes terminate transcription in discrete regions near their poly(A) sites.

We have examined transcription termination of two closely linked Drosophila melanogaster chorion genes, s36-1 and s38-1, using the electron microscope. Our method is unusual and is independent of in vitro nuclear run-on transcription. By measuring transcription unit lengths in chromatin spreads, we can localize efficient termination sites to a region of approximately 210 bp for s36-1 and approximately 365 bp for s38-1. The center of this region is approximately 105 nucleotides downstream of the poly(A) site for the s36-1 gene, and approximately 400 nucleotides downstream for the s38-1 gene. Thus, these two Drosophila chorion genes terminate more closely to their poly(A) addition sites and in a shorter region than many other polyadenylated genes examined to date.

RNP particles at splice junction sequences on Drosophila chorion transcripts.

HnRNP particles are located at specific sites on nascent transcripts when chromatin is spread for electron microscopic visualization. To determine if the sequences bound by the particles play a role in RNA processing, we have correlated the nascent transcript morphology of Drosophila chorion s36-1 and s38-1 genes with their nucleotide sequences. We find that RNP particles about 25 nm in diameter are at the splice junctions of the introns in these two transcripts. On the more mature chorion transcripts, a single larger (40 nm) particle is occasionally seen in the same vicinity, which probably results from the coalescence of the two smaller particles. This RNP structure may be involved in bringing splice junctions into close proximity and in maintaining this proximity during the bipartite splicing intermediate stage.

Novel amplification and transcriptional activity of chorion genes in Drosophila melanogaster follicle cells.

Single-copy chorion genes coding for egg shell proteins are amplified in the follicle cells of Drosophila melanogaster egg chambers. Chromatin spreads of appropriately staged follicle cells reveal complex, multi-forked chromosomal structures in which one chromatin strand branches into two, which themselves branch out, and so on. In one micrograph, 13 strands originating from a single strand were observed. These structures can account for the maximal amplification occurring in the center of the domain, where the major chorion genes are located, and the decreasing amplification of flanking sequences to both sides. The amplification, high transcriptional rate, stage-specific expression, and correlation with known molecular sizes have allowed the putative identification of the single-copy, major chorion genes on the X chromosome and on chromosome III.