Spt16 shows gene-specific influence on Pol III transcription. (A) Spt16 levels are not correlated with transcription from the tRNA genes. Average occupancy profiles of Spt16 on the three groups of tRNA genes classified according to their transcription activity, using the nascent transcription rates (nTRs) of individual genes from the data of Pol III nascentome (Jordán-Pla et al. 2015), kindly provided by A. Jordán-Pla. (B) A typical agarose gel profile of total RNA extracted as detailed in the Materials and Methods section is shown. Total RNA is resolved on the 2.8% agarose gel and stained with ethidium bromide. (C) ChIP-real time PCR measurements of the Pol III (Rpc160-myc) occupancy at some of the genes was made at both permissive and nonpermissive temperatures of growth. Occupancies were calculated by the % input method and normalized with the TELVIR region. Values for the SNR6 (U6 snRNA) TATA were divided by 2 to fit on the scale with others. (D) Measurements of RNA levels at 37°C in the wild-type (W) and spt16-197 (M) cells by northern blotting. Three biological replicates are shown. Pol II-transcribed SNR14 (U4 snRNA) gene was taken as positive control. Arrow marks the pre-tRNA, seen for some of the genes. (E) Quantification of total RNA levels at 37°C by qPCR in the spt16-197 cells normalized to wild-type RNA levels for selected Pol II- (U4 snRNA) and Pol III-transcribed genes. The P-values are (**) 0.0087, (*) 0.022619, (*) 0.011947, (***) 0.00011, and (****) 0.000002 in the order of their appearance on the graph panel.
