yeast cultures were grown in nutrient - rich ypd media ( 1% yeast extract , 2% peptone , and 2% dextrose ) at 30 on a rotary shaker unless otherwise noted .
for induction of autophagy , cells were grown to mid - log phase ( a600 = 0.7 ) in ypd , collected by centrifugation ( 3000 rpm  5 min ) , washed with water , then resuspended in nitrogen starvation media ( 1.7 g / liter yeast nitrogen base without amino acids and without ammonium sulfate , plus 2% dextrose ) . for northern blot analysis of temperature - sensitive mutants ,
cultures were grown at 30 to an od600 of 0.7 and then incubated at 37 for 1 hr before rna extraction .
spei cut atg31ses1 intergenic fragment ( pcr amplified with oligos ddo1281/-1282 , which added an artificial xhoi site ) into bluescript sk+ ( all oligos used are listed in supporting information , table s2 ) .
two - step pcr mutagenesis was performed using pdd1232 as template and t7 and t3 primers with mutagenic primers ( ddo184/-1284 ; ddo183/-1285 , respectively ) to amplify the fragment containing the tdna deletion ; this fragment was then cloned into bluescript sk+ as above to create pdd1233 .
direct - site - directed mutagenesis was performed on pdd1232 to create pdd1248 ( tdna b - box ) , pdd1249 ( tdna b - box mutant ) , pdd1261 ( tdna a - box mutant ) , and pdd1260 ( tdna::ecori  bamhi linker ) , using ddo1391/-1392 , ddo1393/-1394 , ddo1474/-1475 , and ddo1466/-1467 primer sets , respectively .
the b - box mutant had the invariant cytosine and following guanine bases changed to gc , and the a - box mutant scrambled the entire consensus .
plasmids pdd1262 ( flipped orientation of the tdna ) and pdd1272 ( tdna::etc9 ) were created by cloning ecori  bamhi - digested pcr - amplified fragments using ddo1468/-1469 ( flip ) , or ddo1534/-1535 ( etc9 ) , respectively , into ecori 
yeast genomic dna was used as pcr template . plasmid pdd1263 ( tdna::etc4 ) was constructed by directly ligating complementary oligonucleotides ( ddo1489/-1490 ) containing ecori 
yeast strains were generated from wild - type s. cerevisiae w303 - 1a ; genotypes of all strains used and generated in this study are given in table 1 .
parent tdna::ura3 ( ddy46054607 ) strains were created by amplifying ura3 with primers ddo1279/-1280 containing homology to the flanking region of tv(uac)d , and then this dna was transformed into wild - type ddy3 followed by selection of ura+ colonies and pcr identification of homologous recombinants .
linearized tdna mutant plasmids were digested with xhoi and spei , individually transformed into a tdna::ura3 strain , and 5- fluoroorotic acid ( 5-foa ) resistant colonies were isolated .
yeast strains for chromatin immunoprecipitation ( chip ) were created by crossing existing brf13x - flag ( ddy1495 ) and tfc13x - flag ( ddy3860 ) strains to ddy4607 , and then flag - tagged ura+ progeny were backcrossed to each tdna mutant .
all strains are isogenic to s. cerevisiae w303 - 1a except ddy5010 , which is s288c .
the 9x - myc epitope tag was amplified from a trp1 marked cassette ( knop et al .
atg319x - myc trp+ homologous recombinants were identified by pcr ( ddo1419/-1464 ) and confirmed by western blotting . atg31 and
atg8 knockout strains were constructed by standard yeast homologous recombination . for autophagy induction alkaline phosphatase assays ,
pho13 pho860 strains were created by crossing a pho13::ura3pho860::his3 strain ( kindly provided by daniel klionsky ) to wild - type and tdna mutants .
most northern results were verified with three ( but at least two ) independently isolated mutant strains ; table 1 lists only the specific strains shown in the figures .
5-race analysis was performed on rna isolated from the ddy420 brf1 ii.6 mutant using the first choice rlm - race kit ( ambion / life technologies , am1700 ) .
individual clones were sequenced by standard sanger sequencing and mapped to the s. cerevisiae genome on the saccharomyces genome database at http://www.yeastgenome.org ( cherry et al . 2012 ) .
quantitative rt  pcr was performed as follows : first - strand cdna was synthesized from 0.5 g of total rna after dnase treatment ( rq1 dnase , promega m6101 ) .
synthesis was extended from long transcript specific primer ddo1284 using protoscript m - mulv first - strand cdna synthesis kit ( neb e6300s ) .
quantitative reverse transcription pcr ( qrt  pcr ) was performed on 1:4 diluted cdna using primers ddo1606/-1555 and sybr green super mix ( bio - rad 170 - 8882 ) with 60 annealing temperature .
reactions were run and analyzed using a bio - rad myiq as described ( kim et al .
2011 ) and examined by agarose gel electrophoresis to verify that only the predicted pcr products were amplified .
the primers were designed to specifically amplify readthrough transcripts ; the strategy is described and illustrated in figure s2 .
primers ddo1527/-1555 or ddo1576/-1577 were used to amplify desired regions surrounding tv(uac)d . for western analysis ,
yeast minilysates were prepared by glass bead lysis of log - phase cultures directly in lysis buffer ( 50 mm tris ph 7.5 , 1% sds , 5 mm edta , 14.3 mm -mercaptoethanol , 1 mm pmsf , 2 g / ml leupeptin and pepstatin ) .
pellets from 5 ml ypd culture at a600 1.0 were resuspended in 200 l lysis buffer and then vortexed with glass beads at 4 for 10 min .
lysis buffer , 100 l , was added , and the mixture was boiled for 3 min , cooled on ice , and centrifuged at 4 to remove cell debris . to 100 l of clarified minilysate , an equal amount of 2 sds 
page loading buffer was added , and after boiling , 15 l was loaded on 12% acrylamide protein gel .
proteins were transferred to millipore immobilon membrane by semidry transfer and incubated in blotto ( 10 tbs/10% sds/5% dry milk ) for 1 hr .
primary myc antibody ( c - myc 9e10 , santa cruz biotechnology ) , anti - mouse ig - horseradish peroxidase secondary antibody ( ge healthcare ) were used for western analysis . immuno - star western chemiluminescent kit ( bio - rad )
yeast strains were grown in ypd rich media to a600 = 1.0 ( 10 cells / ml ) .
cells were harvested by centrifugation , washed once with distilled water , and resuspended at 10 cells / ml in media lacking nitrogen .
cells were incubated for 6 days at 30 on rotatory shaker , and every other day 200 l of culture dilutions was plated on ypd plates in triplicate .
plates were incubated at 30 for 48 hr and survival rate was obtained by counting resulting colonies .
figure 1a depicts the atg31ses1 locus , showing the location of the tv(uac)d tdna , and the extent of transcripts normally produced from the region . to test the initial hypothesis that tv(uac)d might act as an insulator by preventing promiscuous activation of atg31 by regulatory elements associated with the strong promoter of the neighboring divergent ses1 gene , we created tv(uac)d deleted ( tdna ) mutant strains .
mutation of the tv(uac)d trna gene upstream of atg31 results in readthrough of the intergenic sut467 transcript .
( b ) northern blot analysis of atg31 expression in wild - type and tdna strains reveals the extended transcript . the atg31 coding sequence probe hybridized to rna of 800 bp in wild - type strains ( black arrow ) and to rna of 1200 bp after tdna deletion ( red arrow ) . the sut467 probe hybridized to the predicted 300-bp transcript in wild - type cells ( blue arrow ) and to the same 1200-bp extended transcript in tdna strains .
each pair of lanes contained total rna from independent wild - type and mutant strains .
( c ) b - box deletion ( b - box ) or mutation of the invariant cytosine in the b - box ( b - box mut ) also resulted in extended readthrough transcription .
strains used were : ( b ) ddy4625 and ddy3 ( wild - type ) ; ddy4653 and 4624 ( tdna ) ; ( c ) ddy3 ( wt ) ; ddy4652 ( tdna ) ; ddy4769 ( b - box ) ; and ddy4925 ( b - box mut ) .
northern blot analysis using a probe homologous to the atg31 coding sequence ( figure 1b , left ) showed not only an apparent slight increase in the level of atg31 mrna in the tdna strains ( compared to act1 controls ) , but also an increase in the length of the transcript by 400 nucleotides ( shifting from 800 bases in wild - type strains to 1200 bases ) , with apparent absence of the normal length mrna .
recent tiling array and rna - seq studies have identified widespread pervasive and intergenic transcription in eukaryotic cells , and in yeast this often appears to occur as bidirectional transcription from strong promoters ( neil et al .
inspection of data from these studies indicated that the sut467 intergenic transcript initiates upstream of the ses1 promoter and terminates near the tdna ( figure 1a ) ; therefore , we hypothesized that the extended atg31 transcript in the tdna strain was a readthrough sut467 transcript that interferes with the production of the normal atg31 transcript . to confirm that the extended transcript in tdna strains was due to readthrough of sut467 and not a 3 extension , we repeated the northern analysis using a probe specific for the transcribed sut467 rna sequence ( figure 1b , right ) .
the results showed that this probe hybridized to rna of 300 bases in wild - type strains , consistent with previous annotations of sut467 .
the tdna strains showed a longer 1200-base transcript , the same length as when using the atg31 coding - sequence probe .
we concluded that in the absence of the tdna at this region , sut467 readthrough occurs and interferes with normal atg31 transcription initiation , producing only the observed extended rna . since our gross deletion of the tdna sequence removed 90 bp of chromosome iv , we confirmed this readthrough effect by creating strains that either had only the b - box sequence of the tdna deleted or contained a mutation in the invariant cytosine residue in the b - box .
both of these mutations of the tdna were expected to result in loss of tfiiic binding and inhibition of pol iii complex assembly .
northern blot analysis with either probe shown in figure 1c confirmed that in each of these mutant backgrounds , complete readthrough of sut467 occurred as in the tdna strains .
the slightly shorter transcript observed in the tdna strain compared to the b - box and point mutant strains is also consistent with the long transcript being a readthrough from upstream of the tdna , as this reflects the 90-bp deletion . also observed
were shorter rnas hybridizing to only the sut467 probe , which appear to terminate between the tdna and atg31 .
our previous studies of heterochromatin barrier and insulator function of tdnas have shown that assembled tfiiic alone can block the spread of silencing from the hmr locus and can insulate a uas from a promoter ( simms et al .
2008 ) . to determine which components of the pol iii complex are required to block sut467 progression , we analyzed atg31 transcripts in various temperature - sensitive strains compromised for pol iii complex function and formation when pulsed at the nonpermissive temperature before rna extraction .
temperature - sensitive mutations in rna polymerase iii subunit genes rpc31 and rpc160 affect transcription initiation and elongation , respectively ( dieci et al . 1995 ; thuillier et al . 1995 ) .
these mutants had relatively little effect on the ability of the tdna to block progression of sut467 ( lanes 2 and 3 compared to wild type in lane 1 ) as evidenced by a minimal alteration of the ratio of normal to extended transcripts .
in contrast , mutations in the tfiiib subunit encoding brf1 gene ( brf1-ii.6 and -ii.9 ) that impair interactions of brf1p with tbp ( andrau et al .
1999 ) showed a major shift to the longer extended transcript ( lanes 4 and 5 ) , with relatively little normal length rna .
interestingly , this mutant also showed an intermediate length atg31 transcript that initiates just upstream of the tdna coding sequence ( see 5-race analysis below ) .
( a ) northern analysis of temperature - sensitive mutants of the pol iii complex was performed as in figure 1 , except that each culture was shifted from 30 to 37 for 1 hr prior to rna extraction .
extended atg31 transcripts are most prominent in tfiiib and tfiiic subunit mutants , which also express an intermediate length atg31 transcript .
strains used in lanes 18 were ddy3 ( wt ) ; ddy232 ( rpc31 - 236 ) ; ddy246 ( rpc162112 ) ; ddy416 ( brf1 ii.9 ) ; ddy420 ( brf1 ii.6 ) ; ddy261 ( tfc3 g349e ) ; ddy3 ( wt ) ; and ddy4300 ( tfc6 promoter mutant ) .
5-race was performed to map transcriptional start sites ( tss ) for the various transcripts observed in the northern blot analysis of the brf1 ii.6 mutant .
colored solid boxes represent the range of alternative tss , which were observed in three distinct clusters .
the exact saccharomyces genome database coordinates of all mapped tss are given in figure s1 .
rna isolated from the temperature - sensitive , dna - binding defective tfc3 g349e mutant strain ( lefebvre et al .
1994 ) showed apparent equal amounts of both normal and long atg31 transcripts , with a small relative amount of the intermediate transcript ( figure 2a , lane 6 ) .
a strain harboring a mutation in the tfc6 promoter that results in reduced expression of tfc6p and slow growth ( kleinschmidt et al .
2011 ) showed a similar pattern ( lane 8) , shifted a bit more to the long and intermediate transcripts .
these results demonstrate that loss of tfiiic function also results in readthrough sut467 transcription , but this could be due to loss of tfiiib assembly in the absence of full tfiiic activity . to verify that the transcription start site of our readthrough transcript initiates in the region of the annotated sut467 transcriptional start site ( tss ) and to map the tss of the observed intermediate transcript
, we performed 5-race analysis on rna isolated from the brf1-ii.6 mutant , because it contains all three transcripts as detected by northern blotting .
as shown schematically in figure 2b , 5-race ends that correspond to the annotated atg31 mrna , and within a 94-nucleotide range that overlaps the annotated sut467 tss , were mapped .
the intermediate transcript was found to begin very close to the beginning of the trna coding sequence .
the exact saccharomyces genome database chromosome iv coordinates corresponding to each individually mapped 5-race end are listed in figure s1 . to further assess the mechanistic requirements of each pol iii transcription factor in preventing pol ii readthrough transcription ,
we constructed yeast strains specifically modified at the atg31 upstream tdna locus and analyzed the long vs. short rna phenotypes . inverting the orientation of the tdna had no effect , as no extended atg31 mrna was detected ( figure 3a , lane 1 ) .
mutation of the a - box within the tdna or replacement of the tdna with the etc4 site resulted in only the extended transcript being produced ( figure 3a , lanes 2 and 3 ) .
each of these replacements was expected to bind tfiiic , but not be able to efficiently recruit tfiiib or pol iii .
interestingly , replacing the tdna with the tdna remnant upstream of the tim21 gene , recently referred to as etc9 ( nagarajavel et al .
2013 ) , was sufficient to block readthrough transcription ( figure 3a , lane 4 ) .
this tdna remnant has previously been shown to bind both tfiiic and tfiiib , but not the pol iii enzymatic complex ( guffanti et al .
strains were constructed to recruit the entire pol iii complex , tfiiib and tfiiic , or tfiiic alone to the atg31ses1 intergenic region .
each construct was tested for the ability to block readthrough and for binding of pol iii transcription factor complexes to the ectopic locations .
( a ) schematic of the modified atg31 loci and northern blot of each strain using the atg31 probe .
lane 1 , ddy4816 ( tdna flip ) ; lane 2 , ddy4817 ( a - box mut ) ; lane 3 , ddy4819 ( etc4 replacement ) ; lane 4 , ddy4970 ( etc9 replacement ) ; and lane 5 , ddy4925 ( b - box mut ) .
replacement of the tdna by etc4 , or mutating the a - box or b - box , resulted in the presence of the extended transcript ( red labels ) .
however , inversion of the tdna sequence or replacement with the etc9 sequence still blocked readthrough ( black labels ) .
( b and c ) confirmation of expected pol iii transcription factor binding in the above mutants by chromatin immunoprecipitation .
each tdna mutant strain was crossed to strains containing either brf13x - flag or tfc13x - flag alleles , and then subjected to chip analysis using anti - flag antibody .
( b ) the absence of tfiiib upstream of atg31 in the a - box mutant , etc4 replacement , and b - box mutant correlates with the presence of the extended transcript , suggesting that tfiiib binding is required to block readthrough .
strains used ( left to right ) were ddy4935 , -4943 , -4949 , -5003 , and -4946 .
( c ) chip analysis of brf13xflag and tfc13xflag strains demonstrates that tfiiic but not tfiiib is bound in etc4 replacement strains , indicating that tfiiic binding alone can not block readthrough transcription .
strains used ( left to right ) were ddy4938 , -5003 , -4949 , -3860 , -5006 , and -4917 .
these results suggest that recruitment of tfiiib is the critical step that prevents readthrough transcription of sut467 , as binding of tfiiic alone at the etc4 site is not sufficient to block pol ii progression .
mutation of the a - box has been demonstrated to impair tfiiib assembly ( huibregtse and engelke 1989 ) , and
these interpretations assume that each of these sequences used to replace the tdna have the same in vivo binding characteristics at the atg31 locus as they do in their native chromosomal locations . to confirm such assumptions regarding the presence or absence of each transcription factor complex at these sequences
when moved to the atg31 locus , we crossed a 3x - flag - epitope - tagged brf1 allele into each of these mutants .
chip results using anti - flag antibody shown in figure 3b demonstrate that the tdna flip and etc9 alleles are strongly enriched for tfiiib at levels comparable to wild - type tdnas , while insertions unable to block readthrough transcription ( a - box mut , etc4 , and b - box mut ) had significantly reduced tfiiib chip signals , comparable to background signals observed in the no antibody control panels .
primers amplifying a separate control tdna on chromosome iii showed similar enrichment in each of the samples , indicating that equivalent amounts of chip dna were added to each pcr reaction .
we also created strains containing a 3x - flag - epitope - tagged tfc1 allele to assess the binding of tfiiic at modified atg31 loci .
the results in figure 3c show that tfiiic but not tfiiib is associated with the etc4 insertion , and both tfiiic and tfiiib are bound at the etc9 insertion and at the wild - type tdna locus .
importantly , and contrary to results seen in our earlier tdna heterochromatin blocking studies ( simms et al .
2008 ) , tfiiic binding alone to etc4 is not sufficient to block cryptic transcript readthrough .
previous studies on the heterochromatin barrier activity of tdnas revealed the involvement of other chromatin - associated proteins in this extratranscriptional function ( donze et al .
2007 ) . to assess the potential role of these tdna associated proteins in blocking readthrough transcription , we performed northern blot analysis ( using the atg31 coding sequence probe ) on rna isolated from a number of these mutants .
the results in figure 4a showed that each of these mutants contain mostly normal - length atg31 transcripts ; however , low levels of readthrough are apparent in some strains , most obvious in nhp6 ( lane 3 ) and smc3 ( lane 12 ) mutants in the particular blot shown . however , the intensity of these signals was relatively weak and was often difficult to consistently distinguish from background in different blots .
genetic factors involved in tdna chromatin boundary function have minimal effects on blocking of pol ii progression through tv(uac)d .
( a ) rna from strains containing mutations that weaken tdna boundary function were analyzed by northern blotting using the atg31 probe .
strains in lanes 19 were ddy3 , -947 , -1376 , -2236 , -2058 , -2509 , -1631 , -1676 , and -5010 ; lane 10 , sg154.2 ; lanes 1113 , roy1032 , -1060 , and -1063 ; lane 14 , ddy4925 .
( b ) rt  pcr analysis of readthrough transcription also shows only minimal effects . to confirm these apparent low levels of readthrough
, we used readthrough transcript - specific primers to develop an rt  pcr assay to measure differences in the relative levels of the long transcript compared to a wild - type strain .
the inset in figure 4b shows an inverted ethidium - stained gel image that verifies that the primers specifically amplified the readthrough cdna , as the b - box mutant strain showed significantly higher levels of rt  pcr product than the wild - type strain .
there also appears to be a low level of readthrough in the wild - type strain , which is consistent with a genome - wide transcriptome analysis that identified a single readthrough clone overlapping this locus ( miura et al .
quantitative rt  pcr was performed on the same rna samples shown in the northern blot in figure 4a , and those that showed a significant increase in the long atg31 transcript relative to the wild - type parent are shown in figure 4b . the b - box mutant strain measured 80-fold more readthrough transcript than wild type in this assay , while other mutants were confirmed to have modest ( ranging from 2- to 15-fold ) yet detectable increased levels of the long transcript as suggested by the northern analysis .
given the extended 5-utr present on the long atg31 transcript , we next asked to what extent translation of the atg31 protein was affected by readthrough sut467 transcription .
we created atg319x - myc epitope - tagged strains in wild - type and tdna mutant backgrounds and then analyzed atg31 protein expression by western blotting . in each strain producing the long transcript , we observed a drastic reduction in atg31p levels ( figure 5a ) .
autophagy is a conserved cellular response that recycles cellular components upon nutrient limitation and during normal regulated molecular turnover and involves the formation of autophagosome vesicles that capture and degrade macromolecules after fusion with other membrane bound vesicles ( reggiori and klionsky 2013 ; stanley et al .
( a ) western blot analysis was performed on wild - type and tdna mutants containing an atg319x - myc tag allele to determine whether the extended transcript affects translation of atg31p .
no other bands were observed on the western blot , demonstrating that the extended transcript does not lead to production of an extended polypeptide .
strains used were ddy5012 ( wt ) , ddy5014 ( tdna ) , ddy5018 ( b - box ) , and ddy5020 ( b - box mut ) .
( b ) inhibition of autophagy induction in yeast expressing the extended transcript as measured by the pho860 alkaline phosphatase assay .
strains used were : ddy5051 ( wt control ) , ddy5072 ( tdna ) , ddy5078 ( b - box ) , ddy5081 ( b - box mut ) , ddy5044 ( atg8 ) , and ddy5046 ( atg31 ) .
( c ) production of the extended transcript is associated with reduced survival under nitrogen starvation conditions .
strains used were : ddy5012 ( wt ) , ddy5081 ( b - box mut ) , and ddy4764 ( atg31 ) . since atg31p is required for the formation of autophagosomes upon nitrogen starvation ( kabeya et al .
2007 ) , we tested the efficiency of this response using two well - characterized assays to measure autophagy induction .
we first created a series of strains that produce the readthrough transcript and contain the pho860 and pho13 alleles .
these mutations reduce background alkaline phosphatase levels , and the phosphatase activity of the precursor pho860 protein can be activated only if it is proteolytically processed during autophagy ( klionsky 2007 ) .
since atg31 is required for these events , we reasoned that the reduction in atg31p levels due to the extended 5-utr would result in reduced processing of pho860p upon induction of autophagy and therefore reduced levels of alkaline phosphatase activity upon shifting cells to nitrogen starvation conditions .
figure 5b shows this to be the case , as strains producing the readthrough transcript showed significantly reduced induction of phosphatase activity compared to a pho13 pho860 strain producing only normal atg31 mrna ( wt in figure 5b ) .
complete deletion of atg31 or atg8 in control strains severely reduced starvation - induced phosphatase activity as expected .
these results confirm that induction of autophagy is compromised in strains that predominately produce the long atg31 transcript . as a second assay for the efficiency of autophagy induction , we tested the viability of yeast cells producing the long transcript when placed under autophagy - inducing conditions .
previous studies have shown that complete deletion of atg31 results in reduced survival of cells undergoing nitrogen starvation due to inhibition of autophagy ( kabeya et al .
when we tested a strain containing the b - box mutation in the tdna ( producing the long transcript ) , we found an intermediate level of survival compared with wild - type and atg31 strains ( figure 5c ) .
this loss of survival is not due to effects on the neighboring ses1 gene , as wild - type and tdna mutant strains show equivalent expression levels of ses1 when analyzed by northern blotting ( figure s3 ) .
this result suggests that readthrough of the cryptic transcript reduces atg31p translation to a level that compromises fitness of the cells during nitrogen starvation .
figure 1a depicts the atg31ses1 locus , showing the location of the tv(uac)d tdna , and the extent of transcripts normally produced from the region . to test the initial hypothesis that tv(uac)d might act as an insulator by preventing promiscuous activation of atg31 by regulatory elements associated with the strong promoter of the neighboring divergent ses1 gene , we created tv(uac)d deleted ( tdna ) mutant strains .
mutation of the tv(uac)d trna gene upstream of atg31 results in readthrough of the intergenic sut467 transcript .
( b ) northern blot analysis of atg31 expression in wild - type and tdna strains reveals the extended transcript . the atg31 coding sequence probe hybridized to rna of 800 bp in wild - type strains ( black arrow ) and to rna of 1200 bp after tdna deletion ( red arrow ) . the sut467 probe hybridized to the predicted 300-bp transcript in wild - type cells ( blue arrow ) and to the same 1200-bp extended transcript in tdna strains .
each pair of lanes contained total rna from independent wild - type and mutant strains .
( c ) b - box deletion ( b - box ) or mutation of the invariant cytosine in the b - box ( b - box mut ) also resulted in extended readthrough transcription .
strains used were : ( b ) ddy4625 and ddy3 ( wild - type ) ; ddy4653 and 4624 ( tdna ) ; ( c ) ddy3 ( wt ) ; ddy4652 ( tdna ) ; ddy4769 ( b - box ) ; and ddy4925 ( b - box mut ) .
northern blot analysis using a probe homologous to the atg31 coding sequence ( figure 1b , left ) showed not only an apparent slight increase in the level of atg31 mrna in the tdna strains ( compared to act1 controls ) , but also an increase in the length of the transcript by 400 nucleotides ( shifting from 800 bases in wild - type strains to 1200 bases ) , with apparent absence of the normal length mrna .
recent tiling array and rna - seq studies have identified widespread pervasive and intergenic transcription in eukaryotic cells , and in yeast this often appears to occur as bidirectional transcription from strong promoters ( neil et al .
inspection of data from these studies indicated that the sut467 intergenic transcript initiates upstream of the ses1 promoter and terminates near the tdna ( figure 1a ) ; therefore , we hypothesized that the extended atg31 transcript in the tdna strain was a readthrough sut467 transcript that interferes with the production of the normal atg31 transcript . to confirm that the extended transcript in tdna strains was due to readthrough of sut467 and not a 3 extension , we repeated the northern analysis using a probe specific for the transcribed sut467 rna sequence ( figure 1b , right ) .
the results showed that this probe hybridized to rna of 300 bases in wild - type strains , consistent with previous annotations of sut467 .
the tdna strains showed a longer 1200-base transcript , the same length as when using the atg31 coding - sequence probe .
we concluded that in the absence of the tdna at this region , sut467 readthrough occurs and interferes with normal atg31 transcription initiation , producing only the observed extended rna . since our gross deletion of the tdna sequence removed 90 bp of chromosome iv , we confirmed this readthrough effect by creating strains that either had only the b - box sequence of the tdna deleted or contained a mutation in the invariant cytosine residue in the b - box .
both of these mutations of the tdna were expected to result in loss of tfiiic binding and inhibition of pol iii complex assembly .
northern blot analysis with either probe shown in figure 1c confirmed that in each of these mutant backgrounds , complete readthrough of sut467 occurred as in the tdna strains .
the slightly shorter transcript observed in the tdna strain compared to the b - box and point mutant strains is also consistent with the long transcript being a readthrough from upstream of the tdna , as this reflects the 90-bp deletion . also observed
were shorter rnas hybridizing to only the sut467 probe , which appear to terminate between the tdna and atg31 .
our previous studies of heterochromatin barrier and insulator function of tdnas have shown that assembled tfiiic alone can block the spread of silencing from the hmr locus and can insulate a uas from a promoter ( simms et al .
2008 ) . to determine which components of the pol iii complex are required to block sut467 progression , we analyzed atg31 transcripts in various temperature - sensitive strains compromised for pol iii complex function and formation when pulsed at the nonpermissive temperature before rna extraction
temperature - sensitive mutations in rna polymerase iii subunit genes rpc31 and rpc160 affect transcription initiation and elongation , respectively ( dieci et al . 1995 ; thuillier et al . 1995 ) .
these mutants had relatively little effect on the ability of the tdna to block progression of sut467 ( lanes 2 and 3 compared to wild type in lane 1 ) as evidenced by a minimal alteration of the ratio of normal to extended transcripts .
in contrast , mutations in the tfiiib subunit encoding brf1 gene ( brf1-ii.6 and -ii.9 ) that impair interactions of brf1p with tbp ( andrau et al . 1999 ) showed a major shift to the longer extended transcript ( lanes 4 and 5 ) , with relatively little normal length rna .
interestingly , this mutant also showed an intermediate length atg31 transcript that initiates just upstream of the tdna coding sequence ( see 5-race analysis below ) .
( a ) northern analysis of temperature - sensitive mutants of the pol iii complex was performed as in figure 1 , except that each culture was shifted from 30 to 37 for 1 hr prior to rna extraction .
extended atg31 transcripts are most prominent in tfiiib and tfiiic subunit mutants , which also express an intermediate length atg31 transcript .
strains used in lanes 18 were ddy3 ( wt ) ; ddy232 ( rpc31 - 236 ) ; ddy246 ( rpc162112 ) ; ddy416 ( brf1 ii.9 ) ; ddy420 ( brf1 ii.6 ) ; ddy261 ( tfc3 g349e ) ; ddy3 ( wt ) ; and ddy4300 ( tfc6 promoter mutant ) .
5-race was performed to map transcriptional start sites ( tss ) for the various transcripts observed in the northern blot analysis of the brf1 ii.6 mutant .
colored solid boxes represent the range of alternative tss , which were observed in three distinct clusters .
the exact saccharomyces genome database coordinates of all mapped tss are given in figure s1 .
rna isolated from the temperature - sensitive , dna - binding defective tfc3 g349e mutant strain ( lefebvre et al .
1994 ) showed apparent equal amounts of both normal and long atg31 transcripts , with a small relative amount of the intermediate transcript ( figure 2a , lane 6 ) .
a strain harboring a mutation in the tfc6 promoter that results in reduced expression of tfc6p and slow growth ( kleinschmidt et al .
2011 ) showed a similar pattern ( lane 8) , shifted a bit more to the long and intermediate transcripts .
these results demonstrate that loss of tfiiic function also results in readthrough sut467 transcription , but this could be due to loss of tfiiib assembly in the absence of full tfiiic activity . to verify that the transcription start site of our readthrough transcript initiates in the region of the annotated sut467 transcriptional start site ( tss ) and to map the tss of the observed intermediate transcript
, we performed 5-race analysis on rna isolated from the brf1-ii.6 mutant , because it contains all three transcripts as detected by northern blotting .
as shown schematically in figure 2b , 5-race ends that correspond to the annotated atg31 mrna , and within a 94-nucleotide range that overlaps the annotated sut467 tss , were mapped .
the intermediate transcript was found to begin very close to the beginning of the trna coding sequence .
the exact saccharomyces genome database chromosome iv coordinates corresponding to each individually mapped 5-race end are listed in figure s1 . to further assess the mechanistic requirements of each pol iii transcription factor in preventing pol ii readthrough transcription ,
we constructed yeast strains specifically modified at the atg31 upstream tdna locus and analyzed the long vs. short rna phenotypes . inverting the orientation of the tdna had no effect , as no extended atg31 mrna was detected ( figure 3a , lane 1 ) .
mutation of the a - box within the tdna or replacement of the tdna with the etc4 site resulted in only the extended transcript being produced ( figure 3a , lanes 2 and 3 ) .
each of these replacements was expected to bind tfiiic , but not be able to efficiently recruit tfiiib or pol iii .
interestingly , replacing the tdna with the tdna remnant upstream of the tim21 gene , recently referred to as etc9 ( nagarajavel et al . 2013 ) , was sufficient to block readthrough transcription ( figure 3a , lane 4 ) .
this tdna remnant has previously been shown to bind both tfiiic and tfiiib , but not the pol iii enzymatic complex ( guffanti et al . 2006 ) .
strains were constructed to recruit the entire pol iii complex , tfiiib and tfiiic , or tfiiic alone to the atg31ses1 intergenic region .
each construct was tested for the ability to block readthrough and for binding of pol iii transcription factor complexes to the ectopic locations .
( a ) schematic of the modified atg31 loci and northern blot of each strain using the atg31 probe .
lane 1 , ddy4816 ( tdna flip ) ; lane 2 , ddy4817 ( a - box mut ) ; lane 3 , ddy4819 ( etc4 replacement ) ; lane 4 , ddy4970 ( etc9 replacement ) ; and lane 5 , ddy4925 ( b - box mut ) .
replacement of the tdna by etc4 , or mutating the a - box or b - box , resulted in the presence of the extended transcript ( red labels ) .
however , inversion of the tdna sequence or replacement with the etc9 sequence still blocked readthrough ( black labels ) .
( b and c ) confirmation of expected pol iii transcription factor binding in the above mutants by chromatin immunoprecipitation .
each tdna mutant strain was crossed to strains containing either brf13x - flag or tfc13x - flag alleles , and then subjected to chip analysis using anti - flag antibody .
( b ) the absence of tfiiib upstream of atg31 in the a - box mutant , etc4 replacement , and b - box mutant correlates with the presence of the extended transcript , suggesting that tfiiib binding is required to block readthrough .
strains used ( left to right ) were ddy4935 , -4943 , -4949 , -5003 , and -4946 .
( c ) chip analysis of brf13xflag and tfc13xflag strains demonstrates that tfiiic but not tfiiib is bound in etc4 replacement strains , indicating that tfiiic binding alone can not block readthrough transcription .
strains used ( left to right ) were ddy4938 , -5003 , -4949 , -3860 , -5006 , and -4917 .
these results suggest that recruitment of tfiiib is the critical step that prevents readthrough transcription of sut467 , as binding of tfiiic alone at the etc4 site is not sufficient to block pol ii progression .
mutation of the a - box has been demonstrated to impair tfiiib assembly ( huibregtse and engelke 1989 ) , and this mutation also allows readthrough .
these interpretations assume that each of these sequences used to replace the tdna have the same in vivo binding characteristics at the atg31 locus as they do in their native chromosomal locations . to confirm such assumptions regarding the presence or absence of each transcription factor complex at these sequences
when moved to the atg31 locus , we crossed a 3x - flag - epitope - tagged brf1 allele into each of these mutants .
chip results using anti - flag antibody shown in figure 3b demonstrate that the tdna flip and etc9 alleles are strongly enriched for tfiiib at levels comparable to wild - type tdnas , while insertions unable to block readthrough transcription ( a - box mut , etc4 , and b - box mut ) had significantly reduced tfiiib chip signals , comparable to background signals observed in the no antibody control panels .
primers amplifying a separate control tdna on chromosome iii showed similar enrichment in each of the samples , indicating that equivalent amounts of chip dna were added to each pcr reaction .
we also created strains containing a 3x - flag - epitope - tagged tfc1 allele to assess the binding of tfiiic at modified atg31 loci .
the results in figure 3c show that tfiiic but not tfiiib is associated with the etc4 insertion , and both tfiiic and tfiiib are bound at the etc9 insertion and at the wild - type tdna locus .
importantly , and contrary to results seen in our earlier tdna heterochromatin blocking studies ( simms et al .
2008 ) , tfiiic binding alone to etc4 is not sufficient to block cryptic transcript readthrough .
previous studies on the heterochromatin barrier activity of tdnas revealed the involvement of other chromatin - associated proteins in this extratranscriptional function ( donze et al .
2007 ) . to assess the potential role of these tdna associated proteins in blocking readthrough transcription , we performed northern blot analysis ( using the atg31 coding sequence probe ) on rna isolated from a number of these mutants .
the results in figure 4a showed that each of these mutants contain mostly normal - length atg31 transcripts ; however , low levels of readthrough are apparent in some strains , most obvious in nhp6 ( lane 3 ) and smc3 ( lane 12 ) mutants in the particular blot shown .
however , the intensity of these signals was relatively weak and was often difficult to consistently distinguish from background in different blots .
genetic factors involved in tdna chromatin boundary function have minimal effects on blocking of pol ii progression through tv(uac)d .
( a ) rna from strains containing mutations that weaken tdna boundary function were analyzed by northern blotting using the atg31 probe .
strains in lanes 19 were ddy3 , -947 , -1376 , -2236 , -2058 , -2509 , -1631 , -1676 , and -5010 ; lane 10 , sg154.2 ; lanes 1113 , roy1032 , -1060 , and -1063 ; lane 14 , ddy4925 .
( b ) rt  pcr analysis of readthrough transcription also shows only minimal effects . to confirm these apparent low levels of readthrough
, we used readthrough transcript - specific primers to develop an rt  pcr assay to measure differences in the relative levels of the long transcript compared to a wild - type strain .
the inset in figure 4b shows an inverted ethidium - stained gel image that verifies that the primers specifically amplified the readthrough cdna , as the b - box mutant strain showed significantly higher levels of rt  pcr product than the wild - type strain .
there also appears to be a low level of readthrough in the wild - type strain , which is consistent with a genome - wide transcriptome analysis that identified a single readthrough clone overlapping this locus ( miura et al .
quantitative rt  pcr was performed on the same rna samples shown in the northern blot in figure 4a , and those that showed a significant increase in the long atg31 transcript relative to the wild - type parent are shown in figure 4b .
the b - box mutant strain measured 80-fold more readthrough transcript than wild type in this assay , while other mutants were confirmed to have modest ( ranging from 2- to 15-fold ) yet detectable increased levels of the long transcript as suggested by the northern analysis .
given the extended 5-utr present on the long atg31 transcript , we next asked to what extent translation of the atg31 protein was affected by readthrough sut467 transcription .
we created atg319x - myc epitope - tagged strains in wild - type and tdna mutant backgrounds and then analyzed atg31 protein expression by western blotting . in each strain producing the long transcript , we observed a drastic reduction in atg31p levels ( figure 5a ) .
autophagy is a conserved cellular response that recycles cellular components upon nutrient limitation and during normal regulated molecular turnover and involves the formation of autophagosome vesicles that capture and degrade macromolecules after fusion with other membrane bound vesicles ( reggiori and klionsky 2013 ; stanley et al .
( a ) western blot analysis was performed on wild - type and tdna mutants containing an atg319x - myc tag allele to determine whether the extended transcript affects translation of atg31p .
no other bands were observed on the western blot , demonstrating that the extended transcript does not lead to production of an extended polypeptide .
strains used were ddy5012 ( wt ) , ddy5014 ( tdna ) , ddy5018 ( b - box ) , and ddy5020 ( b - box mut ) .
( b ) inhibition of autophagy induction in yeast expressing the extended transcript as measured by the pho860 alkaline phosphatase assay .
strains used were : ddy5051 ( wt control ) , ddy5072 ( tdna ) , ddy5078 ( b - box ) , ddy5081 ( b - box mut ) , ddy5044 ( atg8 ) , and ddy5046 ( atg31 ) .
( c ) production of the extended transcript is associated with reduced survival under nitrogen starvation conditions .
strains used were : ddy5012 ( wt ) , ddy5081 ( b - box mut ) , and ddy4764 ( atg31 ) . since atg31p is required for the formation of autophagosomes upon nitrogen starvation ( kabeya et al .
2007 ) , we tested the efficiency of this response using two well - characterized assays to measure autophagy induction .
we first created a series of strains that produce the readthrough transcript and contain the pho860 and pho13 alleles .
these mutations reduce background alkaline phosphatase levels , and the phosphatase activity of the precursor pho860 protein can be activated only if it is proteolytically processed during autophagy ( klionsky 2007 ) . since atg31 is required for these events , we reasoned that the reduction in atg31p levels due to the extended 5-utr would result in reduced processing of pho860p upon induction of autophagy and therefore reduced levels of alkaline phosphatase activity upon shifting cells to nitrogen starvation conditions .
figure 5b shows this to be the case , as strains producing the readthrough transcript showed significantly reduced induction of phosphatase activity compared to a pho13 pho860 strain producing only normal atg31 mrna ( wt in figure 5b ) .
complete deletion of atg31 or atg8 in control strains severely reduced starvation - induced phosphatase activity as expected .
these results confirm that induction of autophagy is compromised in strains that predominately produce the long atg31 transcript . as a second assay for the efficiency of autophagy induction , we tested the viability of yeast cells producing the long transcript when placed under autophagy - inducing conditions .
previous studies have shown that complete deletion of atg31 results in reduced survival of cells undergoing nitrogen starvation due to inhibition of autophagy ( kabeya et al .
when we tested a strain containing the b - box mutation in the tdna ( producing the long transcript ) , we found an intermediate level of survival compared with wild - type and atg31 strains ( figure 5c ) .
this loss of survival is not due to effects on the neighboring ses1 gene , as wild - type and tdna mutant strains show equivalent expression levels of ses1 when analyzed by northern blotting ( figure s3 ) .
this result suggests that readthrough of the cryptic transcript reduces atg31p translation to a level that compromises fitness of the cells during nitrogen starvation .
the results described in this study demonstrate that stable rna polymerase iii transcription factor complexes containing tfiiib assembled at tdnas have the capacity to block the progression of intergenic transcription by rna polymerase ii .
high - throughput microarray and sequencing technologies have led to the identification of much more diversity in transcriptomes , from prokaryotes to humans , than was previously appreciated ( core et al .
such pervasive transcripts include the cryptic unstable transcripts ( cuts ) , stable unannotated transcripts ( suts ) , xrn1-sensitive unstable transcripts ( xuts ) , and meiotic unannotated transcripts ( muts ) ( wyers et al .
additionally , alterations in the prevalence of intergenic transcripts and transcript start and end sites have been observed under different growth and stress conditions ( xu et al .
since the vast majority of these cryptic transcripts and transcript isoforms have unknown functions , it has been speculated that they may represent inherent sloppiness of the transcriptional process , referred to as 
transcriptional noise  ( struhl 2007 ) . where functions of such pervasive transcription have been identified in s. cerevisiae
, it appears that it is not necessarily the rna produced but the act of transcription itself that leads to the observed function .
the short noncoding srg1 transcript inhibits ser3 expression by transcriptional interference and promoter occlusion mechanisms , as the path of the srg1 transcript overlaps transcription factor binding sites within the ser3 promoter ( martens et al .
, the srg1 transcript terminates near the beginning of ser3 , while mutation of the tdna at sut467 results in uncontrolled readthrough all the way to the end of atg31 .
similar cis - linked mechanisms may be at work at other yeast loci where noncoding transcription appears to block initiation or elongation of adh1 ( bird et al .
2006 ) , imd2 ( kuehner and brow 2008 ) , ura2 ( thiebaut et al . 2008 ) , flo11 ( bumgarner et al . 2009 ) , pho84 ( camblong et al . 2007 ) , and ime4 ( hongay et al . 2006 ; gelfand et al .
there is evidence that there are also trans - effects of the noncoding rna product regulating the pho84 locus ( camblong et al .
additionally , full repression of yeast gal genes ( houseley et al . 2008 ) , ime1 ( van werven et al . 2012 ) , and again pho84 ( camblong et al . 2007 ) requires chromatin modifications associated with ongoing noncoding transcription .
while more instances are likely yet to be identified , this handful of yeast genes has incorporated intergenic transcription into their regulatory programs and generally appears to use it as a means of repression .
however , we show in this study that if left unchecked , progression of noncoding transcription can have negative consequences on neighboring gene expression , resulting in reduced fitness of cells .
this result demonstrating a cryptic transcript - blocking activity of bound pol iii complexes can be added to the list of extratranscriptional effects of the rna polymerase iii system .
our results presented here demonstrate that the tdna upstream of atg31 protects against such repressive transcriptional interference effects .
our data are consistent with a model in which tfiiib , as part of the pol iii complex associated with the tv(uac)d tdna , serves as a physical impediment to elongating rna pol ii initiating at the sut467 transcriptional start site . in the absence of tfiiib , nearly complete readthrough by pol ii occurs to produce an extended sut467atg31 rna transcript .
this transcript is not efficiently , if at all , translated into atg31 protein , as scanning ribosomes ( kozak 2005 ) attaching at the 5 end of the extended transcript would encounter start and stop codons before reaching the atg31 start codon .
this readthrough transcript appears to be both capped and polyadenylated , as the 5-race protocol includes a phosphatase treatment before decapping and adaptor ligation , ensuring that only capped 5 ends are mapped , and the long transcript is enriched in northern analysis of poly(a)-purified rna ( a. korde , unpublished data ) . the small amount of atg31p we detect in our western blots likely results from low levels of normal atg31 transcripts that are undetectable in northern blots from cells grown in rich media .
extracts from nitrogen - starved tdna mutants show a slight increase in protein levels , along with detectable normal atg31 transcripts in northern blots of rna isolated from the same cultures ( a. korde , unpublished data ) .
this suggests that under conditions that induce atg31 , limited normal initiation is slightly enhanced , but protein levels are still lower than in wild - type cells .
previous work from our lab and others has shown that certain extratranscriptional effects associated with tdnas can be mediated by binding of the tfiiic complex alone .
propagation of silencing at the hmr mating locus can be blocked by replacing the tdna downstream of the hmr - i silencer with an etc site , and insertion of an etc site between uasg and gal10 insulates the promoter from gal4p activation ( simms et al .
heterochromatin boundary activity of tfiiic - only containing complexes is also observed in schizosaccharomyces pombe ( noma et al .
additionally , the etc6 site within the tfc6 promoter may modulate transcription by an insulator - like mechanism ( kleinschmidt et al .
in this case of preventing readthrough of intergenic transcription , the binding of tfiiic alone is clearly not sufficient . while tfiiic binds to b - box sequences in vitro with extremely high affinity ( lefebvre et al .
2003 ) , this binding is somehow tempered by passage of the pol iii enzymatic complex during transcription of the internal control element regions . on the other hand , after recruitment of tfiiib by tfiiic , the tightly bound tfiiib complex appears to be fixed , as in vitro experiments have shown that tfiiib  dna complexes are resistant to high salt and heparin treatments ( kassavetis et al . 1990 ; kassavetis et al . 1995 ) .
the fully assembled tfiiib complex also is thought to be  kinetically trapped  ( cloutier et al . 2001 ) , with a half - life on the order of a full yeast cell cycle , and fully assembled tfiiib likely persists at tdnas until regulated release during mitosis or stationary phase ( fairley et al . 2003 ; roberts et al .
such characteristics of tfiiib are consistent with our results that suggest that formation of this complex is the major impediment to cryptic transcript readthrough by sut467 .
these results are also compatible with earlier in vitro studies that demonstrated the ability of pol iii to transcribe through assembled tfiiic but not assembled tfiiib ( bardeleben et al .
our results suggest that tfiiic yields to pol ii in a similar manner as it does to pol iii , since replacing the tdna with etc4 allowed readthrough of pol ii even though chromatin immunoprecipitation analysis revealed that tfiiic was bound to the ectopic etc4 site ( figure 3c ) . while we have not mapped the exact 3 end of the sut467 transcript , the annotated end mapped by tiling array analysis ( xu et al .
2009 ) places it within 20 bp of the 5 extent of the expected tfiiib footprint at this tdna ( schematically depicted in figure s1 ) .
estimation of this 5 end of the tfiiib footprint is based on earlier in vitro footprinting studies ( kassavetis et al .
1989 ) and a recent global  bootprinting  analysis of in vivo bound pol iii transcription factors ( nagarajavel et al .
the location of the 3 end of sut467 is consistent with tfiiib being a transcriptional roadblock that is resistant to displacement by transcribing pol ii .
a curious sidelight to this study is the appearance of the intermediate length transcript in tfiiib and tfiiic mutants , but not in tdna mutants .
this is most likely initiated by pol ii , as in brf1 , tfc3 , and tfc6 mutants , the tdna terminator sequence is still present , so it is unlikely that this is a pol iii transcript .
we speculate that in these mutants , tfiiib binding still occurs , but is unstable , and perhaps dissociation of the brf1p and bdp1p subunits occurs before loss of tbp at the site .
such a lingering tbp might then recruit factors necessary to then subsequently recruit pol ii immediately upstream of the tdna .
alternatively , the pol iii complex may mask a cryptic pol ii promoter , which is revealed in a subset of cells containing mutations in the pol iii transcription factors .
there is mounting evidence that a much larger fraction of genomes is transcribed than was previously appreciated . while rna degradation pathways generally keep most of these transcripts at low levels ( wolin et al .
2012 ) , it has become clear that the act of intergenic transcription can have significant effects on neighboring genes . due to such observations , one must consider how mutation of a specific genomic locus may affect expression of nearby genes in addition to the targeted gene when assigning the actual cause of observed phenotypes ( wei et al .
iii extratranscriptional effects , we are conducting rna - seq analysis of wild - type vs. 
 tfc6p underexpressing mutant strains .
previous studies have been conducted to determine the global effects of pol iii deficiencies ( conesa et al . 2005 ) , but the rna was analyzed by coding sequence microarray , which could not detect effects involving intergenic transcription .
inspection of preliminary rna - seq results suggests that when the pol iii complex is globally compromised , several tdna proximal genes may be affected as described here , and in other possibly unique ways ( q. wang , a. korde , and c. nowak , unpublished results ) .
this type of result also raises the question of how to interpret phenotypes due to mutations that may globally affect intergenic transcription ( which may be relevant in mutants of other dna and chromatin binding proteins ) , or as shown here for mutation at a specific locus , as unchecked cryptic transcription can lead to unexpected and even detrimental misexpression of downstream genes . while a subset of pervasive transcription products themselves may be noise
 the major function of eukaryotic rna polymerase iii is to transcribe transfer rna , 5s ribosomal rna , and other small non - protein - coding rna molecules . 
 assembly of the rna polymerase iii complex on chromosomal dna requires the sequential binding of transcription factor complexes tfiiic and tfiiib . 
 recent evidence has suggested that in addition to producing rna transcripts , chromatin - assembled rna polymerase iii complexes may mediate additional nuclear functions that include chromatin boundary , nucleosome phasing , and general genome organization activities . 
 this study provides evidence of another such  
 extratranscriptional  activity of assembled rna polymerase iii complexes , which is the ability to block progression of intergenic rna polymerase ii transcription . 
 we demonstrate that the rna polymerase iii complex bound to the trna gene upstream of the saccharomyces cerevisiae atg31 gene protects the atg31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic sut467 transcription unit . 
 this protection is predominately mediated by binding of the tfiiib complex . when tfiiib binding to this trna gene is weakened , an extended sut467atg31 readthrough transcript is produced , resulting in compromised atg31 translation . since the atg31 gene product is required for autophagy , strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy - inducing nitrogen starvation conditions . 
 given the recent discovery of widespread pervasive transcription in all forms of life , protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled rna polymerase iii complexes and possibly other dna binding proteins . 
Materials and Methods
Results
Mutation of the tDNA upstream of 
TFIIIB binding is correlated with blocking of 
Mutations in genes affecting tDNA heterochromatin barrier function have minimal impact on transcript blocking
The extended 
Discussion
Supplementary Material