Our laboratory has used tRNA-mediated suppression to study tRNA biogenesis in S. pombe
. Pol III terminates tRNA gene transcription with high efficiency (~90%) at an oligo(dT) stretch of 5 Ts in S. pombe
and this increases at longer oligo(dT) tracts (9
). Suppression of a premature UGA stop codon in ade6-704
UCA decreases accumulation of red pigment, providing an assay that can also be used for genetic screening. Two types of termination reporters were developed that produce suppressor tRNA from either a dimeric or monomeric tRNA gene construct (9
) (A and B). In the monomeric assay strain yYH1, a 7T terminator resides at the natural position, within a few nucleotides from the 3′-end of the suppressor tRNA sequence (B) (39
). In yYH1, 3′-oligo(U) length of transcripts terminated at T1 is a major determinant of whether the nascent pre-tRNA is processed to a mature functional suppressor-tRNA (reviewed in 40). In the dimeric system assay strain yJI1 (A), a suppressor-tRNA lies downstream of the natural oligo(dT) terminator of the upstream tRNA sequence (9
). WT S. pombe
pol III initiates normally at the upstream tRNASer
and terminates at the oligo(dT) without transcribing the downstream suppressor-tRNA (9
). A suppression phenotype occurs only if pol III fails to terminate at the natural oligo(dT) terminator and transcribes into the suppressor-tRNA which is followed by a 21T failsafe terminator (9
). This was used to isolate mutants in the Rpb9-homologous domain of C11 that cause terminator readthrough (9
). RNA quantification revealed as expected that the mutant pols III read through a 6T terminator more readily than a 7T terminator (9
). To make the dimeric-based screen more sensitive, the 6T terminator was replaced with 5T in strain yKR1. Given that 5T is an efficient terminator in S. pombe
and one-half of all S. pombe
tRNA genes have a 5T terminator, more than any other T length class (41
), this is a physiologically representative reporter (39
Figure 1. (A and B) Schematic showing two types of suppressor tRNA gene reporter constructs used in this study, and corresponding strain names with the number of Ts in the monitored terminators. In (B), the cRT region is highly complementary to the pre-tRNA sequence (more ...)
After isolation from yKR1, the mutant C37 alleles were also assayed for suppression in a second dimeric strain, yJI1, for readthrough of a 6T terminator, and then in the monomeric strain yYH1 for RNA 3′-oligo(U) cleavage function at a 7T terminator from which terminated transcripts are released. This was followed by analysis of the C37 alleles for readthrough transcripts produced in vivo from a monomeric gene with either a 5T or 7T terminator.
Isolation of C37 mutants
Nucleotide analog-based mutagenic PCR was used to construct a randomly mutagenized C37 library (8
) in the pRep4X expression vector. To assess the diversity of the library prior to screening in S. pombe
, we sequenced 40 randomly chosen bacterial clones. These collectively contained 64 substitutions and 3 insertions or deletions. Each of the four nucleotide identities, A, T, G and C underwent transitional and transversional mutagenesis such that each identity was mutated to all three other identities (). We also evaluated the rate at which amino acids were changed. Thirty percent of the 40 clones showed no change in amino acid sequence, 22.5% had one, 27.5% had two, and 15% had more than two amino acid mutations, plus 5% with an insertion or deletion. We plotted the distribution of amino acid mutations in the 40 random clones along the length of the C37 polypeptide (A). We conclude that the library contained a wide variety of mutations randomly distributed throughout the C37 amino acid sequence ( and A).
Figure 2. Location of single point mutants of S. pombe C37 and their terminator readthrough phenotypes. (A) Distribution of amino acid substitutions in the unselected library along the length of C37 (X axis). (B) Distribution of single amino acid substitution mutants (more ...)
We screened ~10 000 colonies for suppression in yKR1, and obtained ~120 suppressed colonies at a rate of ~3.0%. Plasmids from the suppressed colonies were recovered, purified and retransformed into yKR1 (5T terminator) and yJI1 (6T terminator) which verified them. Upon sequencing, 32 contained single amino acid-altering mutations, 43 contained two, 28 contained three or more, 15 contained nonsense mutations alone or in combination with other mutations and 5 contained a single premature stop codon. Some of the single mutation mutants were obtained several times and with different amino acids at the same position (). These data demonstrate a variety of mutations per codon in the library, multi-fold coverage, and near exhaustive screening of the library. Moreover, not a single WT C37 clone was recovered as a suppressed mutant from our library screening even though WT C37 clones were present at ~30% in the library, indicating specificity of the screening.
Single mutation C37 mutants obtained from yKR1 and phenotype in yKR1, yJI1 and yYH1 (see text)
Parallel screening with a similarly mutagenized C53 library that bears mutation frequency comparable with the C37 library produced no suppressed mutants from 140 000 colonies (C53–1, ). In a further attempt to isolate C53 readthrough mutants, we created a second library with a higher mutation frequency (C53–2 in ). Twenty mutants were obtained but these all showed only weak phenotype relative to the C37 mutants and all contained more than one mutation () and were not further characterized. Thus, mutation of C37 much more readily caused readthrough than mutation of C53. We therefore focused on the C37 mutants.
Library screening of Rcp37 and Rpc53 library
It should also be noted that as was the case for prior C11 screening (8
), the present screening as well as further analyses occurred in cells that have a chromosomal copy of WT C37. This strategy has an advantage as it theoretically allows identification of important mutations that when mutated might otherwise be lethal in the absence of the endogenous WT protein. Potential limitations are noted in the Discussion.
C37 mutants cluster in a region previously localized near Rpc2p in the pol III active center
Only 10 amino-acid positions were mutated in the 33 single mutants, represented by 14 unique mutations, and 27 of these were clustered at positions 189 to 201 (B). This distribution is remarkably distinct from the single mutations found throughout C37 in the unselected library (A). Twenty-three of the 33 readthrough mutants were at T191 and T192, replaced by various amino acids, and V195, many of which were independent isolates obtained multiple times ().
After isolation, the mutant alleles were retransformed into reporter strain yKR1, along with WT C37 and empty vector pRep4X, to confirm that their suppression phenotype was due to the C37 allele and not mutation at a chromosomal locus. Different C37 alleles produced different levels of suppression, from mild (pink) to strong (white) as exemplified by L41P and T191I, respectively (C).
Comparison of mutants in strains bearing different length oligo(dT) tracts in the dimeric tRNA reporter is a way to gauge their relative readthrough strength in vivo
). Therefore, to test whether the different levels of suppression seen in yKR1 reflected different readthrough strengths of the C37 alleles, we compared the suppression phenotypes of the 14 unique single mutants in yKR1 and yJI1 strains, which bear 5T and 6T terminators (A), along side a previously characterized terminator-readthrough mutant in Rpc2p, C2–T455I (9
) (C and D). This revealed that the eight mutants that produced strong suppression in yKR1 also produced suppression in yJI1, whereas the mutants with moderate to weak suppression in yKR1 produced less suppression in yJI1, consistent with the stronger test terminator in yJI1 (C and D). Although most of the strong phenotype associated mutations were clustered at positions 189–201, the exception, K69 also produced strong phenotype in the single mutants, K69E and K69N.
promoter driving expression from pRep4X-C37 is repressible by thiamine (43
), and as shown in D suppression was repressed by thiamine (E). This confirmed that suppression was due to the C37 alleles driven by the nmt1
Sequence alignment shows that most mutations mapped beyond the C53–C37 dimerization domain in a conserved region that includes S. cerevisiae
C37 residues previously localized near Rpc2p in the pol III active center (11
) (F). For example, V189 of S. pombe
corresponds to T228 of S. cerevisiae
C37, which was reactive with Rpc2p near the active site (11
We also examined C37 mutant alleles with two mutations after retransforming them into yKR1 (A). This revealed that strong suppression occurred only when a mutation was at K69, T191, T192 or E201, which also cause strong phenotype as single mutants, as well as S190, which was not isolated as a single mutation mutant (A). Of the 43 clones with two mutations, 32 contained one at either K69, S190, T191 and T192 but none contained mutations at two of these positions. Thus, the double mutation mutant data are important because it confirms the clustering pattern observed for single mutation mutants (B).
Figure 3. Multiple mutation mutants also cluster in the C-terminal region. (A) tRNA-mediated suppression phenotype of C37 double substitution mutants after retransformation of the mutated C37 plasmid into yKR1. The top row shows the empty vector pRep4X, C37-WT (more ...)
Two classes of C37 mutants: oligo(dT) readthrough and 3′-oligo(U) lengthening
Five clones with moderate suppression contained only a nonsense mutation, two at Q177 and three at Q220 (). The stop at Q220 would truncate 23 amino acids, similar to S. cerevisiae
C37 bearing a 27 amino acid deletion that yields pol IIIΔ which lacks RNA 3′-cleavage activity and exhibits terminator readthrough (11
). We therefore examined these mutants for suppression in the RNA 3′-cleavage-sensitive strain yYH1. Side-by-side comparison with other mutants in both the RNA 3′-cleavage-sensitive and readthrough-sensitive strains is required to demonstrate the striking contrast with which they partition these phenotypes (A). Q177x and Q220x exhibit moderate suppression in yKR1, none in yJI1 (as expected from moderate phenotype in yKR1), but strong suppression in yYH1, similar to C37–L41P and C11–C102S (A), the latter a RNA 3′-cleavage-deficient mutant (8
). These data suggest that C37–Q177x, -Q220x and -L41P cause RNA 3′-oligo(U) lengthening as in cleavage-deficient C11 mutants (8
Figure 4. C37 mutants contain 3′-oligo(U) lengthened nascent pre-tRNAs. (A) Side-by-side analysis of mutants for terminator readthrough phenotype in yKR1, yJI1 (5T and 6T) as well as 3′-oligo(U) lengthening-associated phenotype in yYH1. A range (more ...)
In striking contrast to the suppression pattern for Q177x, Q220x and L41P, is the C-terminal cluster mutant T191I, which exhibits strong suppression in yKR1 and yJI1 but much less, albeit detectable suppression in yYH1 (A). A similar pattern was observed for C37–V189D (not shown). Adjusting adenine content of the media sensitizes the suppression assay and confirmed that C37–T191I exhibits slightly more suppression than WT in yYH1 but less than L41P (B). These data suggest that the C37–T191I class mutants exhibit more terminator readthrough than 3′-oligo(U) lengthening whereas Q177x, Q220x and L41P exhibit more 3′-oligo(U) lengthening than readthrough. Thus, we isolated two classes of phenotypic C37 mutants. We also note that while C37–T191I and C37–L41P are diametric in these phenotypes, this is in contrast to Rpc2–455I, which reproducibly exhibits high levels of both phenotypes (8
) (A). These data support the idea that these phenotypes reflect associated but distinguishable activities that are associated with pol III termination.
Elongated 3′-oligo(U) in C37–L41P accounts for suppression in the RNA 3′-oligo(U) cleavage-sensitive strain, yYH1
A strong phenotype of C37–L41P in yYH1 while weak in yJI1 is similar to C11–C102S (A), the C11 cleavage mutant that produces 3′ elongated oligo(U) (8
). In yYH1, whose suppressor-tRNA gene requires termination at a 7T terminator, the majority of nascent suppressor pre-tRNAs bear only three or fewer Us on their 3′-ends (8
). tRNA-mediated suppression is increased in the C11–C102S cleavage mutant because increasing length to ≥4Us increases affinity of the pre-tRNAs and thereby their effective competition for a limiting amount of S. pombe
La protein which promotes tRNA maturation (8
). We examined 3′-oligo(U) length of the nascent suppressor pre-tRNAs, with WT and cleavage-deficient C11–C102S as controls. We cloned and sequenced ~40 nascent pre-tRNA cDNAs from each mutant and tabulated the percentage with ≥4 Us at the 3′-end (C). This showed 23% and 47% of nascent pre-tRNA transcripts with ≥4 Us for WT and C11–C102S respectively, in good agreement with previous results (8
). At 38%, C37–L41P was increased relative to WT (C). C37–T191I produced lower percentage of nascent transcripts with ≥4 Us as compared with L41P, consistent with its intermediate suppression phenotype relative to WT and C37–T191I (compare with B). These data support the idea that increase in the fraction of pre-tRNAs with 3′-oligo(U) length of ≥4 Us is accompanied by and correlated with suppression in yYH1 (8
) and suggest that certain C37 mutants may influence 3′-oligo(U) length more than others. This provides novel evidence that the C37 point mutants characterized here may affect pol III active site-mediated RNA 3′-oligo(U) cleavage during termination.
Plotting oligo(U) length distribution as in D revealed that C37–T191I and C37–L41P appeared more similar to WT than to C11–C102S. This showed that C37 mutant pols III produce nascent pre-tRNAs with near normal 3′-oligo(U) length, centered at 3Us, upon termination within a 7T terminator. Previous data indicated that RNA 3′-cleavage occurs during termination (8
). The normal length of RNA 3′-oligo(U) was not necessarily expected for C37–T191I because as a strong readthrough mutant with expected fast elongation it might terminate farther into the oligo(dT) tract than WT pol III thereby producing longer 3′-oligo(U).
RNA 3′-cleavage accompanies transcript release at termination
To evaluate the potential for C11-mediated cleavage in the C37–T191I readthrough mutant, we created C11/C37 double mutants and examined 3′-oligo(U) length of nascent pre-tRNA transcripts released at a normal terminator. For this, we examined nascent intron-containing endogenous pre-tRNAIle
UAU, comparing C37–T191I mutants carrying either WT or cleavage-deficient C11–C102S (E). First, it should be noted that it has been known from multiple studies that the 3′-oligo(U) tracts of nascent pol III transcripts are heterogenous in length (reviewed in 40). C11-mediated 3′-oligo(U) metabolism during termination in the context of C37-WT pol III has been examined before (8
). Pol III containing the cleavage-active WT C11 (C11-WT) produces 3′-oligo(U) tracts that are shortened by 1–2 nt relative to the cleavage-deficient pol III mutant containing C11–C102S (8
). For the present study, the critical comparison is 3′-oligo(U) length in C37–T191I carrying C11-WT versus cleavage-deficient C11–C102S. E shows that the peak of 3′-(U) length of nascent pre-tRNAIle
UAU transcripts from C37–T191I/C11-WT reflects shorter 3′-(U) tracts than in C37–T191I/C11–C102S. Since C11-WT versus C11–C102S is the only variable in this experiment it can be reasonably concluded that the 3′-oligo(U) length difference is attributed to the cleavage activity of C11. Thus, although C37–T191I exhibits increased read through of oligo(dT) terminators, its nascent transcripts are nonetheless subjected to C11-mediated 3′-end shortening when it terminates at a normal oligo(dT) terminator.
Suppressor-tRNA readthrough transcripts in the mutants
It was reassuring that tRNA-mediated suppression could be used to reflect the relative readthrough of terminators of varying T length by the mutants (C versus 2D). However, although S. pombe
and other yeast contain a small number of dimeric tRNA genes similar to our reporter genes in yKR1 and yJI1 (44
), the vast majority are monomeric, with a natural oligo(dT) terminator just downstream of the 3′-end of the tRNA sequence (41
). Therefore, we wanted to confirm readthrough of oligo(dT) terminators using the monomeric tRNA gene. For this, we used northern blotting to detect readthrough transcripts from the monomeric suppressor tRNA allele in yAS76 whose naturally positioned 5T terminator is followed by ~100 bp before a strong 8T downstream terminator (B) (9
). By probing for sequence between the natural and downstream terminators we can detect a specific transcript band and assess readthrough of the monomeric tRNA gene natural terminator (A) (39
). yAS68 (lane 1) is a control with the same monomeric suppressor-tRNA as in yAS76 but with 3T in place of the 5T terminator (B) (9
). yAS99 (lane 2) lacks a suppressor-tRNA gene and serves as negative control (9
C2-T455I (lane 3) is a positive control for readthrough (9
). As expected, yAS99, empty vector, and WT C37 showed only background levels of readthrough transcript (A, lanes 2, 4 and 5). The mutants that exhibit strong suppression in yKR1 showed more readthrough transcripts than the weak readthrough mutant C37–L41P (, compare lanes 6–11 with lane 12).
Figure 5. Detection of 5T terminator readthrough transcripts in C37 mutants by northern blotting. RNAs isolated from various mutants and controls; the same blot was probed sequentially (after stripping previous probe, not shown) as indicated below. Lanes 1 and (more ...)
The data in A and another blot were quantified using U5 RNA (synthesized by pol II) on the same blots (B) as a loading control. We set yAS68 to represent 100% readthrough, intentionally loading less total RNA to maintain linearity of detection (9
). Quantitation is shown in C, reflecting readthrough of a T5 terminator. Most mutants produced more readthrough transcripts than vector or WT, consistent with their readthrough phenotype. The expected exception was C37–L41P, which exhibits relatively weak readthrough phenotype.
Readthrough of endogenous tRNA gene terminators with evidence of gene-specific termination defects
The substantial readthrough transcripts observed from the suppressor-tRNA gene suggested that we might also detect readthrough of natural terminators of endogenous tRNA genes. We surveyed the genome for tRNA genes whose natural terminators were followed by a not too distant downstream tract of ≥5Ts so that we could detect a distinct readthrough band of a predicted size. Probes specific to the regions downstream of the natural terminators detected readthrough transcripts from the three tRNA genes examined, tRNALysCUU, tRNAProCGG and tRNAIleUAU (D–F). As expected, endogenous readthrough transcripts were observed in C37 mutants and the rpc2-mutant but not the controls. Intriguingly, the relative abundance of these transcripts and the RT transcripts in (A) appear to vary in a gene-specific or terminator context-specific manner in different mutants (Discussion). We also uncovered evidence for tRNA gene-specific effects of the termination mutants (D–F). This was observed in the differential pattern of RT transcripts from the four tRNA genes we examined as can be appreciated by comparing the relative amounts of RT transcripts in lanes 3, 7 and 9 of A, D, E and F). In any case, the presence of readthrough transcripts in the mutants but not controls confirmed that the pol III termination mutants are defective at natural endogenous tRNA gene terminators and therefore exhibit generalized widespread pol III termination deficiency.
The termination mutants show no apparent decrease in overall pol III transcription
As noted in the ‘Introduction’ section, pol III can efficiently recycle on in vitro
-assembled transcription complexes to support multiround transcription in a process referred to as facilitated reinitiation (18–23
). In the yeast in vitro
system, facilitated recycling ‘requires termination at the natural termination signal’ (26
) as well as pol III initiation factors and the pol III subunits C11 and C53/37 (12
). According to current understanding, termination deficiency could plausibly affect transcription by two mechanisms; via effects on single round transcription and on facilitated recycling. With a single passage of pol III, readthrough of a natural tRNA terminator should manifest as an increase in readthrough transcript with corresponding decrease in the normally terminated nascent pre-tRNA, independent of reinitiation. A prediction of this effect would be that the ratio of readthrough to primary nascent pre-tRNA transcripts should increase as termination deficiency decreases. By the second mechanism, if termination promotes reinitiation (12
), defects that impair termination may further decrease tRNA transcript output because in growing yeast most of this should be due to reinitiation. Thus, if facilitated recycling is impaired due to termination deficiency, total output should be diminished in a termination mutant relative to WT. In contrast to this would be effects of the first mechanism only, that total output (primary nascent pre-tRNA + readthrough transcript) would not be diminished even in the presence of demonstrable termination deficiency.
We examined nascent intron-containing pre-tRNAs, which because of their rapid processing, reflect differences in pol III transcription rates in vivo
). Northern blots were probed with an intron probe for nascent pre-tRNALys
CUU, used as a standard in several studies (8
). This probe detects three pre-tRNALys
CUU bands, of which the uppermost is the nascent transcript that contains a 5′ leader, a 3′-oligo(U)-containing trailer, and intron, whereas the lower two are processing intermediates (36
) (see right of G). We found no consistent variation between the amounts of readthrough observed in A and nascent pre-tRNALys
CUU levels in the mutants (G). For example, C37–V189D (lane 7) produced more readthrough of tRNALys
CUU than any other C37 mutant (D) but with no significant decrease in the nascent pre-tRNALys
CUU (G) relative to the other C37 mutants, the vector and C37-WT (compare lanes 4, 5 and 7).
We next examined both the naturally terminated nascent pre-tRNA from a 7T terminator and the readthrough transcript that results at a downstream 8T terminator, with a probe that simultaneously detects both on the same blot (A), followed by separate probing of the blot for U5 as loading control (B). Again, we found no consistent variation in the amounts of readthrough and nascent pre-tRNA in the readthrough mutants when compared with C37-WT and vector controls (A). The rpc2-mutant C2-T455I produced a relatively large amount of readthrough but this was not accompanied by decrease in the pre-tRNA as compared with C37-WT and vector controls (lanes 4 and 5) or C-37-E201V which showed less readthrough (lane 11). C37-V195D produced a relatively large amount of readthrough transcript (lane 10) that was not accompanied by decrease in pre-tRNA as compared to the controls (lanes 4 and 5). Likewise, low readthrough levels in C37–E201V (lane 11) was not accompanied by more nascent pre-tRNA.
Figure 6. Assessment of termination mutants for transcription output of single tRNA genes by northern blotting. RNAs isolated from mutants and controls; the same blot was probed sequentially (after stripping previous probe, not shown). Lanes 1 and 2: RNA from positive (more ...)
We next wanted to test whether the ratio of readthrough transcript to primary nascent pre-tRNA transcript would increase as termination deficiency decreases as would be expected from a 5T versus a 7T terminator. A shows transcripts from a tRNA gene 7T terminator, and C from the same gene with a 5T terminator. This revealed a higher ratio of RT to pre-tRNA in C relative to 6A, confirming an expected increase in RT transcript at the expense of nascent pre-tRNA as termination efficiency decreases. Greater readthrough of 5T was also apparent by quantification after control for loading by U5 RNA (E). These data confirmed that our northern blotting approach reassuringly reflected quantitative differences in termination efficiency for the 7T versus 5T terminator.
Surprisingly, quantitation of triplicate samples for total transcripts (RT + pre-tRNA) revealed more in some of the terminator readthrough mutants than in the controls after correction by U5 (F) (see below and the ‘Discussion’ section). Thus, we readily observe relative increase in RT transcript as termination deficiency decreases from a 7T versus 5T terminator as expected on a single tRNA gene, but no decrease in total transcription output in the termination mutants.
14C-Uridine labeling reveals no decrease in tRNA synthesis or growth on minimal media in the pol III termination mutants
Others have used metabolic labeling to demonstrate impaired tRNA synthesis in mutants (57
). We incubated logarithmically growing cells for 5 minutes in 14
C-uridine to label newly synthesized tRNA. No significant difference between WT and C37 mutants was found (A). Quantification of duplicate experiments revealed no significant deficiency of tRNA synthesis in the C37 mutants (B). We also observed no significant difference after pulse chase labeling (not shown).
Figure 7. Characterization of C37 termination mutants by metabolic labeling of tRNA and growth in minimal media. (A) An equal number of logarithmically growing cells were incubated for 5.0 minutes in 14C-uridine, washed and RNA was purified. Left panel shows ethidium (more ...)
To further characterize the mutants, we examined them for growth on minimal media. The C37 mutants exhibited no significant growth deficiency relative to WT (C).