EM Visualization of Transcription by RNA Polymerase II Yvonne N Osheim, Nick J Proudfoot, Ann L Beyer  Molecular Cell  Volume 3, Issue 3, Pages 379-387 (March 1999) DOI: 10.1016/S1097-2765(00)80465-7

Figure 1 Construction of a Xenopus TFIIIA Minigene and Manipulation of Its Poly(A) Signal (A) Diagram showing the structure of the TFIIIA minigene. Hatched boxes denote exons, thick lines denote flanking sequence and introns, while thin line denotes vector sequence. The deletion of exons 3–8 is indicated, as are the sequences of the different poly(A) signals. (B) RNase protection analysis of total RNA extracted from Xenopus oocytes microinjected with the three different TFIIIA minigene plasmids with different poly(A) signals. The RNA probes used are shown on the right hand side. The boxes denote probe RNAs with arrows indicating positions of poly(A) sites. The lines with arrowheads denote transcripts detected, with the expected lengths of the RNAs indicated. The left-hand panel shows the RNase protection data. M denotes marker lanes. With SPA (lanes 1–5), the major product (S) is 10 nt shorter than the predicted position (S*) (lanes 4–5), which may reflect some degree of duplex instability due to the AU richness of the sequence. This major product (S) is not present in a control lane (Co) in which tRNA was used instead of oocyte RNA (lanes 1–3). It should be noted that this same poly(A) signal has been previously shown to work with nearly 100% efficiency in Xenopus oocytes when placed at the end of the Xenopus laevis α-tubulin gene (Plant and Morgan 1998). With MPA, no band is detected at the usual wild-type position (W), but instead, cryptic bands are detected (C), reflecting low-level alternative poly(A) site selection (lanes 6–8). For each construct, the ratio of readthrough transcript band (RT) to poly(A) site band gives a measure of poly(A) site efficiency. Molecular Cell 1999 3, 379-387DOI: (10.1016/S1097-2765(00)80465-7)

Figure 2 Examples of Terminating and Readthrough Plasmid Genes (A) Electron micrograph of a terminating SPA minigene. The curved arrow indicates the start site of transcription and the direction of transcription on the plasmid. The straight arrow points to the approximate position of the poly(A) sequence on the plasmid. Note that transcription proceeds well beyond the poly(A) sequence and that transcripts are full length at the 3′ end of the gene. Bar = 1 μm. (B) Histogram showing the position of the last transcript for terminating MPA, wtPA, and SPA plasmids, represented as the fraction of the plasmid circumference traversed from the start site of transcription and shown in reference to the plasmid map at the bottom. (Hatched box denotes plasmid; black boxes denote exons; lines denote introns or flanking sequences.) The sample corresponds to the mappable plasmids that displayed termination from two frogs. (C) EM of an SPA minigene plasmid exhibiting readthrough transcription. Bar = 1 μm. (D) EM of an MPA minigene plasmid exhibiting readthrough transcription (center of micrograph). Endogenous rDNA gene in the bottom right corner. Bar = 1 μm. (C) and (D) are shown at the same magnification. Note the difference in length of the readthrough transcripts on the two plasmids, presumably due to cotranscriptional cleavage of the SPA transcripts at the strong poly(A) site. Molecular Cell 1999 3, 379-387DOI: (10.1016/S1097-2765(00)80465-7)

Figure 3 Three wtPA Plasmids from the Same Nucleus Exhibiting Template-Specific Patterns of Termination and RNA Cleavage (A) EM, interpretive tracing, and transcript map of a terminating gene with no RNA cleavage. (B) Same for a terminating gene with an intermediate level of RNA cleavage. About 30% (5 of 17) of the transcripts beyond the poly(A) signal have been cleaved. (C) Same for a terminating gene with characteristics of torpedo-type termination. Note that termination is just beyond the poly(A) sequence. Inset shows a higher magnification picture of the portion of the plasmid that includes the 3′ end of the transcription unit (marked with asterisks). RNA transcripts have been overlined with dotted lines so as to better distinguish the DNA template in this region. Arrows indicate polymerases with no or very short transcripts, which are predicted intermediates in torpedo termination. Note also the ill-defined nature of the template between the last polymerases and the resumption of nucleosomal organization. Molecular Cell 1999 3, 379-387DOI: (10.1016/S1097-2765(00)80465-7)

Figure 4 Three SPA Plasmids from the Same Nucleus Exhibiting Template-Specific Patterns of Termination and RNA Cleavage (A) EM, interpretive tracing, and transcript map of a terminating gene with very little RNA cleavage. (B) Same for a terminating gene with an intermediate level of RNA cleavage. About 37% (11 of 30) of the transcripts beyond the poly(A) signal have been cleaved. (C) See the legend to Figure 3C. In the inset, arrows indicate polymerases with no detectable transcripts, and arrowheads indicate polymerases with very short transcripts, both of which are predicted intermediates in torpedo termination. Molecular Cell 1999 3, 379-387DOI: (10.1016/S1097-2765(00)80465-7)