In Vitro Analysis of Elongation and Termination by Mutant RNA Polymerases with Altered Termination Behavior

The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional start site even when the DNA template is supercoiled. The C53/C37 subcomplex also stimulates the formation of an artificially assembled elongation complex from its component DNA and RNA strands. Protein-RNA and protein-DNA photochemical cross-linking analysis places a segment of C53 close to the RNA 3 end and transcribed DNA strand at the catalytic center of the pol III elongation complex. We discuss the implications of these findings for the mechanism of transcriptional termination by pol III and propose a structural as well as functional correspondence between the C53/C37 subcomplex and the RNA polymerase II initiation factor TFIIF. RNA polymerase III (pol III)3 transcribes genes encoding short RNA products that are essential for protein synthesis (e.g. tRNAs, 5 S rRNA), RNA processing (e.g. U6 small nuclear RNA, the RNA subunit of RNase P), protein transport (7SL RNA of the signal recognition particle), and diverse other functions, particularly in higher eukaryotes (1). Pol III is brought to the transcriptional start sites of its genes through interactions with its central 3-subunit initiation factor, TFIIIB (subunits Brf1, TBP, and Bdp1). TFIIIB, in turn, is assembled on DNA upstream of the transcriptional start site in a largely sequenceindependent process by its six-subunit assembly factor, TFIIIC, which binds to two promoter elements (boxA and boxB) located downstream of the transcriptional start site. An additional initiation factor, TFIIIA, serves solely as the adaptor for assembling TFIIIC on 5 S rRNA genes. Vertebrates contain an additional form of TFIIIB in which its paralogue Brf2 replaces Brf1 for transcription of a class of genes that use the SNAP c complex in place of TFIIIC as their TFIIIB-assembly factor (2, 3). Once it has been assembled onto DNA, Saccharomyces cerevisiae TFIIIB suffices for recruiting pol III for robust and accurately initiating transcription in vitro (4); at least in yeast, this also appears to be the case in vivo (5). The complexity of pol III contrasts with the apparent simplicity of its core transcription apparatus (the TFIIIB⅐DNA complex and diverse assembly factors). Five pol III subunits have no counterparts in the considerably less complex pol II (with its merely 12 subunits). Three of these pol III subunits (C82, C34, and C31) form a subassembly that interacts with the TFIIIB⅐DNA complex and is required for specifically initiating transcription (6). Two subunits, C53 and C37, form a subassembly that limits the processivity of pol III elongation and contributes to the abil...

Section: Discussionmentioning

confidence: 99%

The C53/C37 Subcomplex of RNA Polymerase III Lies Near the Active Site and Participates in Promoter Opening

Kassavetis

Prakash²,

Shim

2010

“…Both pausing and the associated hydrolytic RNA cleavage are affected by mutations in the C160, C128, and C11 subunits of yeast Pol III (7)(8)(9). In the case of C128 and C11, the same mutations have also been shown to affect the termination properties of Pol III, thus suggesting a functional interplay between hydrolytic RNA cleavage and transcription termination (9,10). The termination-prone character of Pol III is consistent with class III genes being very short, a fact that reduces the probability of unwanted termination signals within the coding regions.…”

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confidence: 99%

“…4) we then analyzed the positional requirements for T 5 terminator weakening. In all of these constructs, the T 13 element was replaced by T 10 , a sequence that works as efficiently as T 13 as a downstream terminator and does not interfere with the T 5 weakening effect (data not shown). In construct 5, the distance between the T 4 and T 10 stretches, which are separated by only 1 bp in the L_3ЈN template (lane 2), was increased to 11 bp.…”

Section: Oligo(dt) Termination Signals In Different Eukaryoticmentioning

confidence: 99%

Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III

Braglia

Percudani

Dieci

2005

103

112

Eukaryotic RNA polymerase (Pol) III terminates transcription at short runs of T residues in the coding DNA strand. By genomic analysis, we found that T 5 and T 4 are the shortest Pol III termination signals in yeasts and mammals, respectively, and that, at variance with yeast, oligo(dT) terminators longer than T 5 are very rare in mammals. In Saccharomyces cerevisiae, the strength of T 5 as a terminator was found to be largely influenced by both the upstream and the downstream sequence context. In particular, the CT sequence, which is naturally present downstream of T 5 in the 3-flank of some tDNAs, was found to act as a terminator-weakening element that facilitates translocation by reducing Pol III pausing at T 5 . In contrast, tDNA transcription termination was highly efficient when T 5 was followed by an A or G residue. Surprisingly, however, when a terminationproficient T 5 signal was taken out from the tDNA context and placed downstream of a fragment of the SCR1 gene, its termination activity was compromised, both in vitro and in vivo. Even the T 6 sequence, acting as a strong terminator in tRNA gene contexts, was unexpectedly weak within the SNR52 transcription unit, where it naturally occurs. The observed sequence context effects reflect intrinsic recognition properties of Pol III, because they were still observed in a simplified in vitro transcription system only consisting of purified RNA polymerase and template DNA. Our findings strengthen the notion that termination signal recognition by Pol III is influenced in a complex way by the region surrounding the T cluster and suggest that read-through transcription beyond T clusters might play a significant role in the biogenesis of class III gene products.Eukaryotic RNA polymerase (Pol) 1 III is unique among DNAdependent RNA polymerases in recognizing a simple run of T residues on the coding strand as a termination signal, in the apparent absence of accessory factors. The minimal signal thought to be sufficient to provoke Pol III termination varies among different eukaryotes. T 4 suffices for termination by Xenopus and human Pol III (1, 2), T 4 /T 5 for Schizosaccharomyces pombe Pol III (3), and T 5 /T 6 for Saccharomyces cerevisiae Pol III (4). Pol III termination within T clusters is generally heterogeneous and progressive (5), and termination efficiency tends to increase with the length of the T run (4). Mechanistically, Pol III termination involves extensive pausing, dictated by the T cluster itself (5, 6). Even during elongation, the addition of three UMP residues in succession is particularly slow (5). Pol III pausing at T clusters sets cycles of hydrolytic RNA chain retraction (7). Both pausing and the associated hydrolytic RNA cleavage are affected by mutations in the C160, C128, and C11 subunits of yeast Pol III (7-9). In the case of C128 and C11, the same mutations have also been shown to affect the termination properties of Pol III, thus suggesting a functional interplay between hydrolytic RNA cleavage and transcription termination (9, 10). The t...

“…RNA polymerase III preparations isolated from several yeast strains bearing increased or decreased termination mutations have an altered response to several U-rich pause sites within the SUP4 template (11). Because of our previous observation (12) that the amounts of mono-and dinucleotide exonuclease products released concurrently with the transcription reaction are directly proportional to the content of oligo(U) tracts in the transcript, we have characterized the set of ret1 mutations in the second largest pol III subunit for the specific activity and substrate preference of the 3Ј-exonuclease of each mutant pol III.…”

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confidence: 99%

Mutational Analysis of the Hydrolytic Activity of Yeast RNA Polymerase III

Bobkova

Habib

Alexander

et al. 1999

For 25 mutant alleles of ret1, encoding the second largest subunit of yeast RNA polymerase III, we have studied the polymerase III nuclease activity, measuring both the total yield and dinucleotide product composition. Mutations affecting amino acids 309 -325 gave slightly elevated nuclease activity. In region 367-376, two mutations gave 12-15-fold increased nuclease activity. Our results do not support the catalytic role in nuclease activity proposed for the conserved DDRD motif in this region (Shirai, T., and Go, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9056 -9060). Mutations centered on a basic region from amino acids 480 to 490, which aligns with Escherichia coli ␤-subunit sequences between Rif r clusters I and II, produce changes in the relative yields of A-and G-containing dinucleotides. Four such mutant polymerases pause during elongation at GPy sequences and, in addition, have a reduced frequency of termination at T 5 terminator sequences. We propose that the side chains of these mutationally altered amino acids are in direct contact with bases in the RNA-DNA hybrid very near the growing 3-end. Two mutations in domain I near the C terminus produced very large increases in exonuclease activity and strongly increased termination, suggesting that this region also contacts the nascent RNA in the hybrid region.The elongation phase of transcription by DNA-dependent RNA polymerases has long been viewed as a repetitive sequence of choices between two alternative outcomes: addition of another nucleotide to the growing RNA or dissociation of enzyme and RNA from the DNA template, resulting in termination (2). More recently, this interplay was seen to involve the additional options of pausing induced by certain template sequences and resection of transcript 3Ј termini, an exonuclease reaction triggered by both spontaneous pausing and artificially arrested elongation (3, 4).A common element in pausing, termination, and 3Ј-exonuclease action is their tendency to occur preferentially when the elongating polymerase has just incorporated several uridylate residues into RNA. Eukaryotic RNA polymerases II and III undergo spontaneous arrest and long pausing, respectively, at U 2 or longer tracts (5, 6), whereas the sites for pol III 1 transcription termination and Escherichia coli (Rho-independent) transcription termination both occur following long U tracts (7,8).In earlier studies of in vivo termination by yeast RNA polymerase III, we found that genetic alteration of the second largest subunit can change this enzyme's ability to continue transcription downstream of a U 5 tract. Mutant polymerases with increased read-through of an intronic U 5 sequence in the SUP4 UIV allele can efficiently produce biologically active suppressor tRNA Tyr (9, 10). These mutant polymerases also read through the U 5 tract in SUP4⌬94 that is placed at the location of the normal U 7 GU 6 terminator, producing abnormally long pre-tRNAs that are not matured to functional tRNA Tyr . By in vivo mutant screening with these same two SUP4 alleles...