Transcription termination by the eukaryotic RNA polymerase III☆
Aneeshkumar G. Arimbasseria, Keshab Rijala, Richard J. Maraiaa,b,⁎
aIntramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, MD, USA
bCommissioned Corps, U.S. Public Health Service, Rockville, MD, USA
a b s t r a c ta r t i c l ei n f o
Received 5 September 2012
Received in revised form 15 October 2012
Accepted 16 October 2012
Available online 23 October 2012
Intrinsic transcript cleavage
RNA polymerase (pol) III transcribes a multitude of tRNA and 5S rRNA genes as well as other small RNA genes
distributed through the genome. By being sequence-specific, precise and efficient, transcription termination
by pol III not only defines the 3′ end of the nascent RNA which directs subsequent association with the sta-
bilizing La protein, it also prevents transcription into downstream DNA and promotes efficient recycling. Each
of the RNA polymerases appears to have evolved unique mechanisms to initiate the process of termination in
response to different types of termination signals. However, in eukaryotes much less is known about the final
stage of termination, destabilization of the elongation complex with release of the RNA and DNA from the po-
lymerase active center. By comparison to pols I and II, pol III exhibits the most direct coupling of the initial
and final stages of termination, both of which occur at a short oligo(dT) tract on the non-template strand
(dA on the template) of the DNA. While pol III termination is autonomous involving the core subunits C2
and probably C1, it also involves subunits C11, C37 and C53, which act on the pol III catalytic center and ex-
hibit homology to the pol II elongation factor TFIIS and TFIIFα/β respectively. Here we compile knowledge of
pol III termination and associate mutations that affect this process with structural elements of the polymerase
that illustrate the importance of C53/37 both at its docking site on the pol III lobe and in the active center. The
models suggest that some of these features may apply to the other eukaryotic pols. This article is part of a
Special Issue entitled: Transcription by Odd Pols.
Published by Elsevier B.V.
Transcription of a DNA template into a complementary RNA is a
most fundamental process of cellular life. Bacterial and archaeal
cells each use a single RNA polymerase (pol) for transcription that
are evolutionarily related to the eukaryotic pols I, II and III (plants
also have pols IV and V, variants of pol II) [1,2]. Pol I transcribes a sin-
gle gene type, the rRNA genes, while pol II transcribes the thousands
to tens of thousands of protein coding and noncoding genes that vary
over orders of magnitude in transcription output, controlled by a vast
combinatorial set of promoters and enhancers. Pol III transcribes
hundreds of tRNA genes which bear similar promoters, as well as 5S
rRNA, U6 snRNA and several other noncoding RNA genes.
Transcription involves three steps: (1) initiation, recruitment of the
bonds; (2) elongation, processive synthesis of the RNA chain, albeit with
intermittent pausing in some cases, and 3) termination, cessation of
RNA synthesis and dissociation of the three components: nascent RNA,
polymerase, and DNA. While each of the eukaryotic pols distinguish
these steps, their specialization appears to include differences in how
the steps are executed, the relative time spent at each step, and how the
steps may be linked to each other. For example, termination and
reinitiation by pol III have been shown to be mechanistically linked
[3–5]. Also, while the three pols use highly similar mechanisms of tran-
scription initiation, there is much more complexity in assembling pol II
initiation complexes than pols I and III . Differences in elongation in-
clude the time spent in this mode, with pol III differing most since at
75–300 nt, its transcripts are relatively short . Another difference is
that pol II uses pausing as a control point, regulated by the positive
transcription elongation factor-b (P-TEFb) .
Transcription termination must involve destabilization of the elon-
gation complex followed by release of the nascent RNA and polymerase.
This is a big transition because elongation complexes must be very sta-
ble in order to avoid the deleterious effects of premature termination
. Failure to terminate can interfere with downstream genes, produce
3′ extended RNAs with potential adverse effects, and deplete the pool of
polymerase that should be available for regulated initiation .
2. Terminators signal the initiation of a two-stage process
Different RNA polymerases use different mechanisms to direct ter-
mination (Fig. 1A–E). Each responds to a specific signal, a terminator
element in the DNA or the elongating RNA that prompts the
Biochimica et Biophysica Acta 1829 (2013) 318–330
☆ This article is part of a Special Issue entitled: Transcription by Odd Pols.
⁎ Corresponding author at: 31 Center Drive, Rm 2A25, Bethesda, MD 20892–2426,
USA. Tel.: +1 301 402 3567.
E-mail address: firstname.lastname@example.org (R.J. Maraia).
1874-9399/$ – see front matter. Published by Elsevier B.V.
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagrm
beginning of the termination process . The process initiates with
recognition of a termination signal and this is followed by induced
cessation of RNA synthesis and release. Some terminators work at a
distance; recognition of the termination signal by the polymerase or
a trans-acting factor is the first stage and this leads, albeit more di-
rectly for some than others, to the second stage, destabilization of a
paused complex with release of the DNA and RNA from the polymer-
ase active center.
Bacterial RNA polymerase can use two kinds of terminators,
factor-dependent and intrinsic . Rho factor is a helicase that rec-
ognizes the nascent RNA at a C-rich sequence after it emerges from
the polymerase and propels along in a 5′–3′ direction to catch up
with the elongation complex, inducing destabilization and termina-
tion (Fig. 1A) . Intrinsic terminators work more directly, i.e., with-
in a relatively short, albeit bipartite terminator element, to coordinate
pausing and destabilization. This involves formation of a hairpin
structure in the transcribed RNA followed by transcription of
oligo(dA)-rich sequence to produce an RNA with an oligo(U)-rich 3′
end [12,14]. Termination occurs as the oligo(rU:dA) hybrid is melted
in the active center of the polymerase (Fig. 1B).
Pol II termination is more complex, involving post-translational
modifications of the polymerase as well as association of a number
of trans-acting factors [10,15]. Similar to bacteria different termina-
tion signals distinguish at least two types of pol II termination mech-
anisms, for poly(A)-containing mRNAs and for poly(A)-independent
small nuclear RNAs . For the first type, the AAUAAA poly(A) addi-
tion site in the elongating RNA recruits a complex of factors that
endonucleolytically cleave the transcript which is then further
processed to become a mRNA. This is followed by 5′–3′ exonucleolytic
digestion of the segment of RNA still attached to the polymerase
(Fig. 1C). Thus for most pol II-transcribed mRNA genes the termina-
tion signal is the poly(A) addition site in the newly synthesized
RNA which acts at a distance; its recognition by RNA-binding factors
initiates a process that ends in termination at a downstream site.
Pol I uses a termination factor, Ydr026C/Nsi1, bound to a specific
element on the downstream DNA  as well RNA endonucleolytic
cleavage followed by 5′ exonucleolytic digestion [18–20]. Pol I tran-
script release occurs within an oligo(dA) tract on the template DNA
 (Fig. 1D). According to a current ‘torpedo’ models of termination
by pols I and II, the 5′–3′ exonucleases catch up with the elongating
polymerases to induce pausing and destabilization leading to the
final stage, release, reminiscent of Rho-dependent termination
Pol III has a most direct acting termination signal, more similar to
but distinct from intrinsic termination by bacterial RNA polymerase,
than pols I or II. Oligo(dA) on the template DNA is sufficient to com-
mence and complete all steps leading to termination by pol III
(Fig. 1E) [22–24]. The pol III enzyme incorporates five stably associat-
ed subunits, the heterotrimer C31/34/82 and heterodimer C37/53 as
two subcomplexes with homology to the pol II ancillary factors,
TFIIE and TFIIF, as well as C11, a two-domain polypeptide with
homology to Rpb9 and the elongation factor TFIIS, that collectively
promote efficient initiation, termination and reinitiation (Fig. 2A
and B) [25–30]. Unlike initiation by pol III which can be directed by
gene types that differ in promoter structure and the trans-acting fac-
tors that recognize them, termination appears to occur by the same
basic mechanism for all (with exceptions that likely reflect variations
on the oligo(dT) theme; see below). These characteristics make pol III
an attractive model for the study of the mechanism of termination by
a eukaryotic RNA polymerase.
In summary, the eukaryotic RNA polymerases use different
signals to initiate the termination process. It is much less clear as
to how their active centers work to execute the final stage of termi-
nation, release from the RNA and DNA by their active centers, and to
what extent they may share mechanisms involved in this stage of
3. Termination by pol III: The signal is oligo(dT)
Comparison of the 3′ oligo(U) sequence at the ends of 5S rRNA
with the 3′ regions of Xenopus 5S rRNA genes suggested that a stretch
of 4 or more T residues in the DNA could act as a pol III terminator
[31–34]. It was shown that pol III in Xenopus oocytes or in solution
would terminate at oligo(dT) at the 3′ ends of 5S rRNA genes [22,24].
While independent studies confirmed oligo(dT) as a universal ter-
minator for pol III, further analysis revealed species-specificity in the
minimum length of the oligo(dT) tract required for termination. For
vertebrates, as few as 4Ts can act as an efficient terminator while
for yeasts variably longer T tracts are required [22,24,35]. In general,
S. cerevisiae requires 6 or more Ts [36–38], while S. pombe requires
5 or more Ts, for efficient (~90%) termination , and this is
supported by genome-wide analyses of tRNA genes [38,39]. It was
further suggested that fission yeast Schizosaccharomyces japonicus re-
quires only 4Ts at a large number of tRNA gene terminators . It is
expected that these differences are manifested in sequence dispar-
ities in the termination-relevant regions of the pol III subunits of
these species. Noteworthy is that the minimal T length requirement
of the different species correlates with the α-amanitin sensitivities
of their pol III , the implications of which will be discussed in a
3.1. Oligo(dT) flanking sequence context effects
Early studies revealed that the sequence surrounding the
oligo(dT) signal can influence termination efficiency [22,24]. This ef-
fect may be most pronounced for vertebrate pol III for oligo(dT)
length of 4 . 4Ts flanked by AA is very inefficient as a terminator,
and similar to Xenopus, most human pol III will read through, whereas
replacing AA with GC increases termination efficiency dramatically
. By contrast, a 5T tract is highly efficient for pol III termination
[22,24] and relatively insensitive to flanking sequence . Nonethe-
less, a 4T terminator flanked by GC can be as efficient as a 5T termina-
tor . This context-dependency of 4T tracts is critical for some pol
III-transcribed genes with 4Ts in their coding region, such as all lysine
tRNA genes and adenovirus VA RNAII. One may suspect that the
necessity for four contiguous Ts in lysine tRNA genes (the invariant
U33 in the mature tRNA followed by the UUU anticodon at positions
34–37) may have set an ancient limit of no fewer than 5Ts as the min-
imal pol III terminator which was later adjusted in multicellular eu-
karyotes by the ability to utilize sequence context effects. In S.
cerevisiae a 5T stretch is relatively inefficient and can act as a termina-
tor only in the right context , although the magnitude of the effect
of flanking sequence may be less than on a 4T terminator for human
pol III. However, there are no hard ‘universal’ rules with regard to
flanking sequence. In S. cerevisiae, CT following the T5 tract can weak-
en the terminator while A or G strengthens it . Also, termination
efficiency of a Xenopus lysine tRNA gene with a 4T terminator was
influenced by flanking sequence differently than for Xenopus 5S
rRNA genes . Notably, these sequence context effects do not re-
quire other factors as pol III itself from yeast or Xenopus exhibited
context-dependent termination [24,38].
Flanking sequence effects might suggest the involvement of sec-
ondary structure. Although proximal hairpins appeared to promote
pol III termination in one study , others indicate no such effects,
confirming oligo(dT) alone as the major terminator element for pol
III. Later in this review we note that for some genes, flanking se-
quence effects may extend to binding sites for extraneous factors
that influence termination, e.g., the NF1 site adjacent to the termina-
tor of the VA1 RNA gene .
While oligo(dT) is clearly the most prevalent terminator for pol III,
isolated reports cite non-canonical pol III terminators, and although
most of these are interrupted oligo(dT) tracts, one was a run of A resi-
dues downstream of a mouse 5S rRNA gene . Human Pol III was
A.G. Arimbasseri et al. / Biochimica et Biophysica Acta 1829 (2013) 318–330
Fig. 1. Schematic of termination mechanisms by multisubunit RNA polymerases. The mechanisms reveal three themes common to more than one pol, (i) 5′–3′ exonuclease diges-
tion of a downstream fragment of the nascent transcript attached to the polymerase, (ii) weakly base pairing oligo(rU:dA) hybrid and (iii) involvement of a helicase, Rho or Sen1.
(A) Rho mediated termination by bacterial RNA polymerase. Rho helicase binds to the nascent transcript at a C-rich region and travels along the RNA in a 5′ to 3′ direction toward
the polymerase to induce destabilization and termination. (B) Intrinsic termination by bacterial RNA polymerase. A hairpin formed by the nascent RNA followed by a T-rich stretch
on non-template strand comprises the intrinsic termination signal. Transcript release occurs within the T stretch. (C) Pol II termination mechanism for poly(A)-containing
mRNA-coding genes. Transcript is cleaved downstream of the poly(A) addition site by cleavage factors associated with the pol II CTD. The polymerase-attached RNA fragment is
digested by Rat1/Xrn2 in 5′–3′ direction. Sen1 helicase also binds to pol II and is required for termination. This figure is oversimplified to emphasize similarities with other pols.
Also, there are different pathways for termination at different classes of genes (see text). (D) Eukaryotic pol I termination mechanism. Most of the transcripts are terminated up-
stream of the Nsi1 (Ydr026C) binding site within the T stretch. Cotranscriptional cleavage of the transcript followed by processive 5′–3′ cleavage by Rat1 is also involved in termi-
nation. (E) Pol III termination mechanism. A stretch of Ts on the non-template strand is sufficient to direct termination.
A.G. Arimbasseri et al. / Biochimica et Biophysica Acta 1829 (2013) 318–330
reported to terminate 3′ of an Alu repeat which did not have 4 or more
consecutive T residues and the potential for RNA hairpin formation was
noted . However mapping pol III terminators using cellular extract
can be complicated. Following in vitro transcription of a B1-Alu gene
element using cellular extract, 3′–5′ digestion of the nascent RNA was
so robust that mapping of the terminator that gave rise to the barely
detectable primary transcript required rapid pulse-chase condi-
tions . Remarkably, this robust 3′ digestion occurred following
transcription of the B1-Alu gene that had a 4T terminator but not the
same gene with a 5T terminator, and this was later attributed to the
3′-protective activity of the 3′ oligo(U) length-specific RNA-binding
protein, La . Another case is the avian adenovirus CELO VA RNA
gene; TTATT caused inefficient termination . Termination within a
T stretch shorter than 4Ts or an interrupted T stretch has been noted
for a human tRNAmetigene .
As noted above and discussed later in more detail, a large number
of human tRNA genes that use non-canonical terminators have been
catalogued but these are variations of oligo(dT), generally T3, T2 or
T1 stretches separated by another nucleotide (interrupted T5 or T4)
. These studies together with the effects of flanking sequence on
termination efficiencies suggest a complex mechanism(s) that
control the efficiency of pol III termination on short oligo(dT)
3.2. Primary and secondary oligo(dT) terminators
Bacteria use a variety of anti-termination mechanisms to control
the polar inclusion or exclusion of segments of polycistronic mRNAs
. An intriguing concept for pol III is that of secondary terminators,
i.e., an oligo(dT) stretch downstream of a weak primary terminator
that can be used to produce a unique longer transcript. In one scenar-
io, a fraction of pol III would terminate at the first oligo(dT), while the
rest would read through to terminate at the downstream terminator
[22,33,49]. Human adenoviral VA RNAIand avian adenovirus CELO
Fig. 2. RNA polymerase III subunits and structural elements that affect termination. (A) Comparison of subunit compositions of pols II and III. The termination subcomplex that
shows homology to TFIIFα/β subunits, the initiation subcomplex that shows similarity to TFIIEα/β, and C11 which is homologous to RPB9 at its N terminus and to the
C-terminal motif of TFIIS at its C terminus, are highlighted. (B) Cartoon of surface features of pol III derived from the electron microscopic structure of pol III (EMD-1802) deposited
by . Blue color reflects the proposed position of the C53/37 dimerization domains and pink shows the initiation subcomplex . The cartoon of the RNA:DNA template hybrid
was obtained from pol II elongation structure (PDB ID: 3HOV). The non-template strand is not shown. Yellow color reflects localization of the N-terminal, Rpb9-homologous domain
of C11 only. Other features discussed in the text and/or elsewhere in the figure are indicated. For other structural representations, refer to ; for additional models see Figs. 3 and
5 in . (C) Cartoon depicting the structural elements that are involved in transcription elongation and termination. The catalytic site, along with the hybrid-binding region and
other elements such as the fork loops, the trigger loop and bridge helix, is considered as part of a larger, extended active center.
A.G. Arimbasseri et al. / Biochimica et Biophysica Acta 1829 (2013) 318–330
VA RNA genes provide examples that can produce two different RNAs
by this mechanism [41,51].
Recent identification of biological functions for tRNA 3′ fragments
derived from the 3′ trailer sequences of pre-tRNAs suggests that partial
read through of the first terminator may be used to generate such tRNA
3′ fragments [52–54, reviewed in 55]. In accord with this, Orioli et al.
identified a large number of tRNA genes in humans with weak termi-
nators followed by a strong secondary terminator . A fraction of
pol III terminates at the weaker terminators while the rest read
through. These observations further suggest the exciting possibility
that pol III termination may be modulated to produce a subclass of
noncoding RNAs, although this remains to be determined. A genome-
wide chromatin immunoprecipitation (ChIP) followed by sequence
analysis of human pol III showed that a significant fraction of pol III
was unexpectedly found to be accumulated somewhat downstream
of the tRNA gene terminators which may reflect among other things
terminator read-through and/or a state of pausing .
4. The termination mechanism
In simple accordance with a kinetic coupling model in which ter-
mination efficiency is inversely related to elongation rate, pausing
provides a window of time during which alterations can occur in
the polymerase that lead to transcript release [56,57]. Indeed, RNA
polymerase pausing at a terminator is prerequisite for termination
[23,58–60]. Kinetic coupling is supported by the fact that methods
that slow elongation lead to increased termination efficiency .
While oligo(dT)-rich tracts are recognized as pause sites, they are
not sufficient to cause termination by other RNA polymerases which
must traverse many kilobases (for pol II megabases) that often con-
tain oligo(dT) by chance or necessity. The bacterial polymerase termi-
nates when an oligo(dT)-rich tract closely follows a hairpin in the
transcript. This arrangement increases specificity by enriching the
signal with more complexity than oligo(dT) and allows both
elements to function toward destabilization of the complex within
the confines of the active center and adjacent elements .
Multiple hypotheses may explain the mechanism(s) of action of
the hairpin during intrinsic termination. A forward translocation
model suggests that hairpin formation induces polymerase to translo-
cate forward without nucleotide incorporation, leading to shortening
and destabilization of the hybrid and transcript release . A second
model suggests that the hairpin induces melting of the proximal end
of the hybrid and destabilization . A third suggests an allosteric
mechanism with direct interaction of the hybrid with the polymerase
trigger loop inducing structural changes that loosen the grip on the
hybrid, hybrid melting and destabilization of the complex .
4.1. Termination must overcome the stability of the elongation complex
One of the most important features of efficient transcription is the
very high degree of processivity of RNA polymerases, without which
there would be an overwhelming number of incomplete transcripts
[9,63]. Poor processivity would not only be wasteful but probably
also cause havoc to RNA processing systems. Thus by design RNA
polymerases must be processive and this is achieved via highly stable
grips on the RNA and the DNA. Part of this stability is rooted in the
complementary base pairing interactions that hold together the tran-
scribed RNA and the DNA template within the active center of the po-
lymerase [64,65]. Elegant studies of bacterial RNA polymerase and pol
II have shown that the nascent RNA forms an 8–9 nt hybrid with the
template DNA [64,65]. A strong RNA:DNA hybrid is one of the primary
elements that is critical to the remarkable stability of the elongation
complex and its processivity [65–67]. Thus the termination process
must destabilize a very stable elongation complex, and it must do it
precisely on cue and only on cue. As reviewed below it appears that
many if not all RNA pols take advantage of a similar characteristic to
do so, the outstanding weakness of rU:dA hybrid base pairs .
4.2. The weak rU:dA hybrid is likely an underlying component of pol III
Oligo(dT) (oligo(dA) in the template) constitutes a part of the ter-
mination complexes of bacterial, phage, archaeal, eukaryotic, and
viral RNA polymerases [11,12,21,69,70]. The demonstration that a
(rU:dA)5hybrid is at least 200 times less stable than the correspond-
ing hybrid containing (rA:dT)5or other sequences led to the proposal
that a decrease in the stability of the hybrid as it acquires rU:dA rich-
ness may be a driving force for termination . A weak hybrid such
as oligo(rU:dA) causes alterations of the elongation complex such as
pausing followed by backtracking [64,71,72]. Shortening is an alter-
nate means of hybrid weakening and this is also implicated in tran-
script release by bacterial RNA polymerase .
As alluded to above, pol III termination appears to be somewhat
similar to intrinsic termination by bacterial RNA polymerase. Intrinsic
terminators are bipartite, with a 7-to-8 nt T-rich tract preceded by a
G+C-rich dyad repeat that forms a stem-loop hairpin in the nascent
RNA upstream of the 3′ U-rich tract [59,61,73–76]. While an encounter
of either a T-rich tract or a G+C-rich dyad may in some contexts cause
pausing, both are required for complete termination . Yet only an
oligo(dT) tract (dA in the template) is required for pol III termination,
but not a dyad repeat, hairpin, or other cis-element. This suggests
that pol III may be exquisitely sensitive to the destabilizing effects of
an oligo(rU:dA) hybrid, a feature that could be accommodated by a po-
lymerase whose substrate genes are short enough as to not require in-
ternal T tracts. The challenge is to understand the mechanisms that
provide pol III the ability to respond so dramatically and efficiently to
a simple signal that induces both pausing and destabilization.
Although there is no direct evidence that rU:dA hybrid weakness
provides a mechanism of pol III termination, some observations sup-
port this. Replacement of rU with Bromo-rU, which forms a more sta-
ble hybrid with dA, in in vitro transcription reactions promotes
terminator read through . Lower reaction temperature, which
among other things increases hybrid stability, increases terminator
read through . Also, the template strand oligo(dA) is required
for termination, while the non-template oligo(dT) can be mutated
with little effect (A.G.A. and R.M., in preparation).
The exact position at which pol III releases its RNA from within the
oligo(dA) tract is heterogeneous as reflected by a variable number of
3′ Us on the RNAs released from a single gene [57,63,77]. A positive
correlation between oligo(U) length and transcript release can be
discerned , consistent with the idea that longer rU:dA hybrids de-
stabilize the elongation complex leading to better termination.
These observations support the idea that an extensive rU:dA hy-
brid destabilizes the elongation complex and promotes termination.
However, none of them exclude the possibility that a contributing in-
fluence may be due to sequence-specific recognition of the rU:dA hy-
brid with allosteric alteration of pol III as an underlying mechanism.
4.3. Is backtracking involved in pol III termination?
A common response of RNA polymerases upon encountering
oligo(dT) is pausing, followed by a more stable block to elongation
caused by backtracking, a process in which the polymerase ‘backs-up’
i.e., slides upstream while maintaining a ~10 bp RNA:DNA hybrid in
the active center [64,71]. During backtracking the RNA 3′ end is
displaced from the catalytic site and ejected out the secondary chan-
nel of the polymerase. RNA synthesis can be restarted from this state
after the catalytic center is converted to a endonucleolytic transcript
cleavage site by the cleavage factor TFIIS for pol II and the GreB and
GreA factors for bacterial RNAP [78–80]. However, polymerase
backtracking that is unresolved by transcript cleavage remains in a
A.G. Arimbasseri et al. / Biochimica et Biophysica Acta 1829 (2013) 318–330
state of transcription arrest. It was proposed that backtracking may be
involved in termination by pol III . Certainly pol III undergoes 3′
retraction, i.e., the cleavage of 3′ terminal residues from RNA in
some cases allowing otherwise stalled complexes to move forward
[25,82–84]. Whether pol III undergoes backtracking as part of termi-
nation is an outstanding question.
5. Pol III subunits involved in termination
The 17 subunits of pol III are intricately connected via an extensive
interaction network . Experimental data indicate that several pol
III subunits can affect the termination process, C2, C11, C37 and C53,
and possibly C1 and others [25,26,29,86–89]. In addition, there have
been multiple reports that extraneous factors can also affect termina-
tion by pol III (below). A challenge is to determine the mechanisms
used by these subunits and extraneous factors to affect termination
and to distinguish between direct and indirect (e.g., allosteric effects
via other subunits) effects.
5.1. The pol III active center is organized from parts of several subunits
For the purposes of this review we shall refer to the active center
as that which contains the catalytic site and its multiple structural
elements such as bridge helix, trigger loop and fork loops that
comprise the catalytic site responsible for nucleotide selection and
phosphodiester bond formation, as well as the adjacent RNA:DNA hy-
brid of nearly 10 bp (Fig. 2C). By comparison of electron micrographs
of pol III with crystal structures of pol II it seems clear that the core
structures of these are conserved (Fig. 2A). Indeed C1 and C2 are
most homologous to their pol II homologs along the catalytic center
including the invariant NADFDGD motif in the largest subunits of all
multisubunit RNA polymerases, and surrounding regions [90,91].
Thus we can be reasonably sure that RPC2, the second largest subunit
of pol III together with the largest subunit C1, forms an extensive
active center similar to but with distinctive differences from pol II
[6,28,91–93]. Certainly, the core pol II structure fits very well into
the electron micrograph envelope of pol III (Fig. 2B) [6,28,91,93].
Peripheral surfaces of pol III such as the jaws and lobe are in prox-
imity to incoming DNA as polymerase moves along the template
(Fig. 2B) . While the dimerization domains of the C37 and C53
polypeptides have been localized to a bulge comprising part of the
upper jaw adjacent to the lobe, biochemical and physical evidence in-
dicate that other parts of these proteins extend into the catalytic site.
A region downstream of the dimerization domain of S. cerevisiae C37
was localized by physical proximity methods near the active site in
vitro  and the homologous region of S. pombe C37 is a hot spot
for mutations that impair termination in vivo . In addition, a region
of C11 with strong homology to elongation factor TFIIS likely inserts
into the active site to mediate intrinsic transcript cleavage with effects
on RNA 3′ end formation during termination [25,26,29,88]. Part of C53
lies close to the RNA 3′ end in the catalytic center of the pol III elonga-
tion complex . These observations suggest that the pol III active
center is a busy place during termination, comprised of parts of multi-
ple subunits. As might be expected for an extended active center and
peripheral nucleic acid-interacting motifs, more than one region of
the C1 and C2 subunits would also be involved in termination.
5.2. The pol III lobe is involved in termination probably via C53/37 and
A major advance toward understanding pol III termination came
from a genome-wide screen in S. cerevisiae to identify genes that af-
fect termination that led to isolation of ret1-1, a transcription mutant
with a mutated allele of RPC2 [86,96]. ret1-1 was selected as a mutant
whose pol III could read through an otherwise functional oligo(dT)
terminator placed within the intron of a suppressor tRNA gene .
Three regions of RPC2 were then chosen for random mutagenesis
and selection for either gain or loss of function, yielding alleles with
mutations that either increased or decreased termination in vivo
and in vitro . Further analyses revealed that many of these C2
termination-altering mutations also increased or decreased the in-
trinsic RNA 3′ cleavage activity of pol III, suggesting a natural relation-
ship between termination and RNA 3′ cleavage .
The three regions of RPC2 were chosen based on termination mu-
tants in the second largest subunit of E. coli RNA polymerase . Of
particular interest is the E. coli polymerase β500-575 which overlaps
with the RPC2 455–524 region and E. coli β1230-1342 region that
overlaps with the RPC2 1061–1081 . Termination-altering muta-
tions in similar regions of bacterial and eukaryotic RNA polymerases
indicate crucial roles in termination in general [87,97] and imply
that all multisubunit RNA polymerases may undergo similar changes
during transition from elongation to termination.
Although the 3-dimensional locations of the three pol III regions
were unknown at the time of their analyses, presently available struc-
tures of S. cerevisiae pol II and pol III allow their localization to three
conserved elements referred to as the lobe, fork loops and anchor re-
gions [87,90] (Fig. 2B). Mutations in lobe residues 300–325 of
S. cerevisiae RPC2 are generally associated with decreased termina-
tion as is the loss of function mutant, ret1-1: T311K . Curiously,
a strong gain of function mutation in this region is K310T, suggesting
that threonines at either 310 or 311 promote termination.
An unbiased genetic screen of zebra fish for digestive system dis-
ruption uncovered a deletion in C2 corresponding to aa 259–300 in S.
cerevisiae C2 that caused a developmental malformation referred to
as the sjm (slimJim) mutant . Recapitulating this deletion in
S. pombe C2 led to dissociation of C11 from pol III, confirming that
this region is required for efficient C11–C2 interaction  (Fig. 2)
[also see Fig. 5 in 29]. Most remarkably, overexpression of zebra fish
C11 reversed the gross digestive anomaly, attributing the phenotype
caused by the C2 mutation to decreased association of C11 and C2
. Since association of C11 is also required (in yeast) for stable asso-
ciation of the C53/37 heterodimer , the sjm mutation may further
compromise the zebra fish pol III. This study, which used molecular
modeling based on comparisons of yeast pols II and III, strongly sup-
ports the idea that the RPC2–RPC11 interface is similar to the RPB2–
RPB9 interface and functionally conserved from yeast to vertebrates
. In addition, sjm was the first to reveal that mutations in a pol III
subunit, a housekeeping enzyme, can have devastating tissue-specific
phenotypic effects , including in humans [99–102].
Therefore the sjm deletion, corresponding to ScRPC2 259–300,
together with the ScRPC2 300–325 mutations identified by Shaaban
et al.  form a contiguous region extending from a C11-interacting
surface to what appears to be the upper ridge of the pol III lobe adja-
cent to the cleft from which incoming DNA moves into the active cen-
ter (Fig. 2B, also see Fig. 5C–G in . This region likely affects the
association of C11 and the C53/37 termination subcomplex with pol III.
In remarkably good agreement with the structural localization of
C53/37 dimerization domains on pol III are physical proximity data
(photo-crosslinking and Fe-BABE mediated cleavage) . The lobe
region of C2 that harbors termination mutations was extensively
cross-linked to the dimerization domains of C53/37 including numer-
ous interactions between C37 and C2 303–329, as well as between
C53 and C2 276–280 , suggesting the lobe as docking site for
C53/37 adjacent to the C2 259–300 region that interacts with the
Rpb9-homologous domain of C11 (see ).
5.3. The fork loops of C2 in the pol III active center are involved in
A large number of ret1 mutations occurred in a conserved region
of C2 encompassing fork loop 2, the majority of which reduced termi-
nation  (Fig. 2C). The fork loops are mobile elements that play
A.G. Arimbasseri et al. / Biochimica et Biophysica Acta 1829 (2013) 318–330
critical roles in RNA strand separation and maintenance of the tran-
scription bubble [103,104]. Deletion of fork loop 2 from pol II is
known to affect the rate of catalysis by impairing sequestration of
substrate NTPs and to cause increased pausing and elongation arrest
The pol II fork loop 2 is a very highly conserved “IGRDGKLA” motif
spanning aa 502–509 of S. cerevisiae RPB2. Structural studies of pol II
had shown that the tip of the loop, containing RDGK, is mobile and
was captured in two conformations, interacting with either the bridge
helix or the non-template DNA [107,108]. The positively charged side
chains at the positions occupied by Arg (R) and Lys (K) are conserved
by all multisubunit RNA polymerases and are suggested to be in-
volved together with an invariant Arg 512, in DNA strand separation
as well as NTP binding and sequestration . The corresponding re-
gion, “FEKT/SRKVS” spanning aa 477–484 of S. cerevisiae C2 is highly
conserved in pol III but divergent from pol II. Mutations of E478 of
S. cerevisiae C2, which is conserved by pol III, affect termination
[57,87]. Substitution with Lys increased while Asp decreased termina-
The region between fork loops 1 and 2 has an imperfect tripeptide
repeat in which every third amino acid is hydrophobic. Most muta-
tions to these hydrophobic residues led to increased termination,
suggesting that a hydrophobic network in this region contributes to
maintaining stability of the elongation complex [57,87].
Another advance in pol III termination was the physical mapping
of a region of C37 to the fork loops and other elements near the cata-
lytic site . Evidence that this was relevant to termination came
from transcription analysis of a mutated C37 with a 5-amino acid
tract deletion (scC37 226–230) surrounding the fork loop and
(βDloopII) interaction site . Pol III reconstituted with the mutated
C37 read through the SUP4 tRNA gene terminator significantly more
than did the nonmutated enzyme .
5.4. The anchor region of C2
All mutations in the anchor region of C2 led to increased termina-
tion. The anchor region of pol II connects the RNA:DNA hybrid-
binding domain to the clamp and lies between flexible switch do-
mains 3 and 4 that control the mobility of the clamp  (Fig. 2B).
The role of the anchor region in pol III termination is currently
unknown although it is expected to control allosteric alterations.
Results of in vitro transcription with the C2 mutants generally
adhered to the kinetic coupling model; faster elongation rate was as-
sociated with reduced termination and slower elongation was associ-
ated with increased termination [57,87], although some exceptions
are noteworthy. Very interesting is the C2 double mutant T455I,
E478K. As a single mutation, T455I shows increased elongation,
decreased termination and efficient RNA release [57,87]. Not surpris-
ingly, the equivalent S. pombe mutant in rpc2-T455I also exhibits
decreased termination [29,30]. The single E478K mutant has slow
elongation with increased terminator recognition (i.e., pausing in
the terminator)  but poor release of RNA . When these muta-
tions were combined as in T455I, E478K, the net effect was decreased
elongation and decreased termination, apparently uncoupling the
kinetics of elongation rate and termination. In this case uncoupling
occurred in association with decreased RNA release, reminiscent of
the effects of nonreleased RNA on the uncoupling of pausing and
termination . However, these two cases are different; for E478
mutants, the pause was more like an arrest. In any case, according
to the model, E478 would be in fork loop 2 while T455 would flank
fork loop 1, in close proximity to the RNA backbone of the RNA:DNA
hybrid. Another mutant that uncouples elongation rate and termina-
tion is C2 K512N . The corresponding pol II residue shows exten-
sive interactions with others in the region, suggesting an intricate
interaction network . So, a mutation here might affect the integ-
rity of the region leading to the termination defect.
In summary, it is remarkable that nearly 15 years after mapping
these S. cerevisiae C2 mutants, the same regions were identified as
in close proximity to the C53/37 termination subcomplex. The lobe
region shows extensive crosslinks with the dimerization domains of
C53/37, suggesting the lobe as a docking site of C53/37 adjacent to
C11. The C-terminal region of C37 was cross-linked to the fork loop
region where another set of mutations were observed. Careful analy-
sis of the C2 mutants revealed that most mutations in the lobe pro-
duced reduced termination which now is most likely due to
decreased association of C53/37. Mutations in the region 455–524
that encompass fork loop 2 led to either increased or decreased ter-
mination, reflecting a pivotal role in setting a balance between elon-
gation and termination. Mutations in the anchor region produced
only increased termination, suggesting that mutations in this region
could be destabilizing the elongation complex.
5.5. C1 and potential effects of α-amanitin on pol III termination
α-Amanitin is a fungal cyclic octapeptide that inhibits transcrip-
tion by eukaryotic RNA polymerases to varying extents [110,111]. It
was noted that α-amanitin termination by Xenopus pol III although
this remains unpublished . Co-crystallization with pol II has
shown that α-amanitin is bound via interactions with the bridge
helix and trigger loop, trapping the latter and limiting its mobility
[112,113]. While pol II is typically most sensitive to amanitin, pol III
shows species-specific sensitivities. S. cerevisiae pol III is quite resis-
tant, vertebrate pol III is more sensitive and S. pombe pol III shows in-
termediate sensitivity . Intriguingly, these amanitin sensitivity
patterns correlate with the oligo(dT) minimal length requirement
for termination: human pol III is most sensitive and requires fewer
Ts, S. cerevisiae pol III is least sensitive and requires the longest T
tract, and S. pombe is intermediate in both amanitin sensitivity and
T length . This correlation suggests that the sequence differences
between different pol III species in the bridge helix and trigger loop
might contribute to termination. These elements are involved in for-
ward translocation of polymerase and thus constitute motifs that de-
termine elongation rate [113–115]. Mutations in this region of C1
affect pausing, RNA cleavage and transcriptional transitions .
Random mutagenesis followed by in vivo screening of this region of
C1 for termination mutants might reveal further insight into mecha-
nisms of termination by pol III.
5.6. C53/C37: A dynamic duo
Another major advance in pol III termination came from charac-
terization of a C11 mutant that produced pol III enzyme devoid of
C11 as well as C53 and C37, known as pol IIIΔ [25,88]. It was shown
that the C53 and C37 subunits are required for oligo(dT) terminator
recognition (pausing) and form a stable heterodimer whose interac-
tion domains are attached to the surface of pol III near the leading
edge of incoming DNA close to the Rpb9-homologous domain of
C11, [28,88,91,92,94]. The C53/37 dimerization domains are homolo-
gous to the dimerization domains of transcription initiation factor
TFIIFβ/α which appear to occupy a similar surface on pol II [27,94].
While these domains hold C53/37 to a surface location, biochemical
evidence indicates that other parts of these polypeptides reach into
the pol III active center.
Association of C53/37 with pol III is dependent on C11. Purified pol
III that lacks C53/37 and C11 fails to terminate at some terminators
and also lacks the intrinsic transcript 3′ cleavage activity . Add
back experiments show that recombinant C11 restored cleavage ac-
tivity but not termination whereas C53/37 restored terminator recog-
nition [25,88]. Kinetic analysis suggests that C53/37 reduces the
elongation rate of pol III on the SUP4 tRNA gene such that pol IIIΔ ap-
pears resistant to several gene-internal pause sites . This
suggested a mechanism by which C53/37 promotes termination, by
A.G. Arimbasseri et al. / Biochimica et Biophysica Acta 1829 (2013) 318–330
reducing elongation rate thereby increasing the pause or residence
time of pol III on the terminator, consistent with kinetic coupling. In
accordance with this model, reducing the pol IIIΔ elongation rate by
decreasing the NTP concentration corrects the termination defect
Although this model is nicely supported by experimental data,
there are some notable points. First, the decreased elongation rate
caused by C53/37 is manifested as pausing throughout the gene not
only at the terminator . Thus, while reduced elongation rate is
consequential to termination according to kinetic coupling, it is not
specific to the terminator. Another indication that C53/37 action is
not specific to termination is evidence that it can function in initiation
by participating in open complex formation similar to certain tran-
scription initiation factors including its pol II homolog, TFIIFα/β
[94,95]. Thus, C53/37 appears to be quite dynamic in the breadth of
its activities. Moreover, related to both this activity and termination
is the requirement for C53/37 along with C11 for facilitated
reinitiation by pol III, which is mechanistically dependent on termina-
tion [3,4,88,117]. Facilitated reinitiation dependent on termination
has also been observed for mammalian pol III [40,44,48,118–121].
Facilitated reinitiation, which has been observed in vitro, is a most
appealing process because it can account for the extraordinary
efficiency with which pol III and the TFIIIB/C stable transcription com-
plexes can be recycled to produce the large amounts of tRNAs, 5S
rRNA and other components required for cellular proliferation
[122,123]. A recent study examined for the first time a potential link
between termination deficiency and overall transcription output.
The results showed that termination deficiency was not accompanied
by a decrease in transcription output and question the degree to
which if any, a link between termination and recycling observed in
vitro is operational in vivo . More studies will be needed to
address the mechanisms by which pol III is able to reinitiate with
apparent high efficiency in vivo.
Photo cross-linking and other physical methods revealed key in-
teractions between C37 and C2 as well as other subunits . As
noted above the dimerization domains of C53 and C37 reside near
the lobe domain of C2 and the Rpb9-homologous region of C11 (Fig.
2B) [28,92,94]. Residues 226–230 in the C-terminal region of C37 far
downstream of its dimerization domain were found to react in the
immediate vicinity of C2 fork loops 1 and 2, βDloopII and a
hybrid-binding region in the active center of pol III . Deletion of
this tract from C37 led to deficiency of pol III termination in vitro .
In support of function of the C-terminal region of C37 in termina-
tion are point mutations in multiple residues in a homologous region
of S. pombe C37 mutants isolated from a genetic termination screen
. The mutants produced increased terminator read-through tran-
scripts from various tRNA genes in vivo . The observations that
C37 residues localize with C2 fork loops and that C2 fork loop muta-
tions had been independently isolated as termination mutants argue
that these regions participate in termination. Moreover, proximity
of these regions of C2 and C37 to the pol III active site further suggests
that termination involves alterations to catalytic activity that are
more complex than passive cessation of phosphodiester bond forma-
tion  (below).
The cumulative data suggest that C37 contributes to and modulates
several polypeptides that can access the catalytic site of an RNA poly-
merase to affect its activity, the prototypical examples of which are
TFIIS for pol II and its bacterial counterpart GreB [80,124]. However,
while TFIIS and presumably the homologous cleavage-domain of C11
access the catalytic center through the secondary channel [80,107],
the path of the C37 and C53 polypeptides to the catalytic center may
be via a more frontal approach perhaps through the DNA cleft region
(Fig. 2B) , although this remains to be determined.
Physical proximity data indicate that the association of the C37
C-region with the RNA:DNA hybrid-binding region of C2 is more
robust in the elongation complex than in the preinitiation complex
. Proximity of the C37 C-region to the βDloopII of C2 was also in-
creased in the elongation complex. The corresponding loop in the pol
II model is part of ‘fraying site II’ which interacts with the frayed, i.e.,
non-annealed, 3′ terminal nucleotide of the RNA . Bioinformatics
suggested a possible structural homology between C53 and MLE, an
RNA helicase . Since C53 can interact with the 3′ end of the RNA
in the elongation complex, the data suggest that it may participate
in RNA:DNA hybrid melting, thereby contributing to destabilization
and termination .
Finally it should be noted that while lack of C37/53 leads to an in-
creased elongation rate, it does not render pol IIIΔ completely incapa-
ble of recognition of the SUP4 tRNA bipartite gene terminator .
This suggests that there is a C37/53/11-independent mode of termi-
nation intrinsic to the core pol III enzyme that is enhanced by C53/
37 (A.A. and R.M., in preparation).
5.7. C11 involvement in intrinsic RNA 3′ cleavage, termination and
As first reported, C11 was believed to be directly involved in
termination because  it was not known at that time that C11 me-
diates association of C53/37 with pol III . C11 is a short polypep-
tide of 110 aa first identified as having strong homology to the pol II
elongation factor TFIIS and responsible for the previously recognized
robust intrinsic transcript cleavage activity of pol III [25,82]. It has
two Zn ribbon motifs separated by what may be a flexible linker.
The N-terminal Zn ribbon of C11 is homologous to the N-terminal
Zn ribbon of subunit RPB9 of pol II and the C-terminal Zn ribbon is
highly homologous to the Zn ribbon of the pol II RNA 3′ cleavage fac-
tor, TFIIS . Similar to TFIIS, the C11 Zn ribbon has the same two
acidic residues at the tip of its loop that presumably position Mg2+
within the pol III catalytic center similar to the homologous residues
of TFIIS in pol II [80,107]. The N-terminal domain of RPB9 is attached
to the lobe domain of second largest subunit RPB2 forming a structure
called the jaw of the polymerase . As noted, the Rpb9-
homologous domain of C11 (yellow in Fig. 2B) occupies a similar po-
sition on the C2 lobe of pol III and is required for association of C53/37
with pol III [28,88,91,94].
Mutations to the C- and N-terminal motifs of C11 have different
effects on termination: RNA 3′ oligo(U) nibbling and prevention of
terminator read through [26,29]. Although the latter may be due to
allosteric effects on C53/37, the cumulative data nonetheless provide
clear evidence that C11 is positioned to affect pol III termination
[25,29,88]. As detailed below, the RNA 3′ cleavage activity of C11 is
active during termination [26,29], although the extent of its role in
the termination process is unresolved. A challenge is to understand
how the two domains of C11 work with each other and with C53/37
both at their surface locations and via their domains that approach
the catalytic center of pol III during termination.
3′ Terminal U residues are preferred substrates of C11-mediated
cleavage by pol III [82,84]. An enticing model would be that shorten-
ing of the rU:dA hybrid by cleavage-mediated removal of 3′ terminal
or frayed Us would lead to hybrid shortening, weakening and destabi-
lization of the complex. However, this model was not supported by
mutations in C11 that compromise RNA cleavage activity since, al-
though these mutations do lead to increased length of the 3′ oligo(U)
tract of the nascent RNA by 1–2 nts both in vivo and in vitro, this was
not accompanied by increased terminator read through [26,29]. The
data suggest that the cleavage activity of C11 mediates 3′ U cleavage
during termination but not termination efficiency per se.
In striking contrast to the C-terminal mutants, a cluster of
N-terminal C11 mutants exhibited increased terminator read through
but not 3′ oligo(U) lengthening . The N-terminal mutations of C11
may affect termination via effects on C53/37 .
A.G. Arimbasseri et al. / Biochimica et Biophysica Acta 1829 (2013) 318–330
Far more C11 C-terminal domain mutants were isolated that im-
paired RNA cleavage than were N-terminal domain mutants that im-
paired terminator recognition, despite their isolation from the same
library [26,29]. Using the same screening approach applied to C37,
the opposite was true; far more C37 mutants were isolated that im-
paired terminator recognition than 3′ oligo(U) lengthening . The
same approach yielded even fewer C53 mutants using both the termi-
nator recognition and cleavage-sensitive screens . These results
provide evidence that of the C11, C53 and C37 subunits, terminator
recognition was by far most sensitive to C37 .
It is noteworthy that C11 exhibits cleavage-dependent and
cleavage-independent activities in pol III termination and associated
processes. While C11 is required for facilitated recycling along with
C53/37, a point mutant that disables its cleavage activity is nonethe-
less as active as the wild type C11 for facilitated recycling .
6. Extraneous factors may affect pol III termination
Several trans-acting factors were shown to augment pol III termi-
nation in vitro. Among them La protein ranks first by historical per-
spective and because of its appealing specificity, affinity for the 3′
oligo(U) tracts of pol III-terminated transcripts.
La exhibits sequence- and length-specific binding to oligo(U),
with the 3′-OH group contributing substantially . These features
along with early findings that La is physically associated with all
newly synthesized pol III transcripts in vivo made it a likely candidate
for involvement in the termination process [127–131]. Experimental
data obtained from in vitro assays supported that La is a transcription
termination, transcript release and reinitiation factor for pol III
[40,48,77,118,120,132,133], although such activities have been
contested [134–136]. Only the non-phosphorylated fraction of
human La was transcriptionally active in vitro, while the phosphory-
lated fraction was inactive . Consistent with this, non-
phosphorylated La is associated with pol III-transcribed genes in
vivo, while phosphorylated La is not . Yet phosphorylated La is
found associated with nascent pre-tRNAs and other pol III transcripts
in HeLa cells, whereas non-phosphorylated La was not . Al-
though in vivo deletion of the La-homologous protein (Lhp1) from
S. cerevisiae caused no decrease in pol III transcription levels ,
it led to increased accumulation of pol III on 5S rRNA genes in vivo,
consistent with a role as a limiting factor for pol III termination
. Thus, although by 3′ oligo(U) binding La protein provides a
link between termination by pol III and processing of its transcripts,
a role in the termination process per se and potential effects on
recycling remain questionable. The mechanistic details of the link be-
tween pol III termination and RNA processing, and its implications,
are described in a later section.
6.1. Other factors reported to promote pol III termination
These include transcription factor IIIC, topoisomerase-1 and PC4.
Both topo-1 and PC4 copurify with TFIIIC and enhance the TFIIIC foot-
print on the downstream promoter . Yeast TFIIIC subunit TFC6
can be cross-linked to the terminators of tRNA and 5S rRNA genes,
suggesting a role in termination [141,142]. In mammalian systems a
TFIIIC-associated activity caused a specific footprint on the terminator
[143,144]. Genetic mutagenesis-based screens of S. pombe that
yielded C11 and C37 mutants that impair terminator recognition
and/or 3′ oligo(U) length [26,29,30] yielded no mutants for similarly
mutagenized sfc6+, the S. pombe homolog of TFC6 and mammalian
TFIIICβ  (K.R. and R.J.M., unpublished observation). Thus, to
date, there is no data linking TFC6 or its counterparts to a functional
role in termination.
The intriguing means by which NF1 promotes pol III termination
of VA1 gene transcription may provide a clue into a mechanism by
which some extraneous factors may operate . Effects of NF1 on
VA1 appear to be gene specific because a NF1 sequence-specific bind-
ing site is found adjacent to the pol III terminator of the VA1 but not
other pol III-transcribed genes . Consistent with this, ChIP analy-
sis did not show preferred localization of NF1 to pol III-transcribed
genes . It is plausible that NF1 bound to terminator-adjacent
DNA acts as a roadblock that induces pausing at the terminator there-
by promoting termination. Such a roadblock may also alter pol III con-
firmation by allosteric means upon collision. Consistent with this, a
roadblock caused by a peptide nucleic acid was shown to increase ter-
mination from a suboptimal terminator .
6.2. A role for chromatin in pol III termination
Although several studies show active involvement of chromatin in
the control of pol III transcription [147–151], little is known about a
role in termination. tRNA genes are generally nucleosome free
. Genome-wide ChIP that focused on the yeast histone variant
H2A.Z revealed a large number of tRNA genes flanked by nucleosomes
. A recent study revealed that the dynamics of a nucleosome that
abuts the terminator of the SUP4 tRNA gene in S. cerevisiae can mod-
ulate the expression level of the tRNA under different condi-
tions . Genome-wide pol III ChIP showed a significant fraction
of pol III accumulation just beyond the terminators of tRNA and
other class III genes, which may reflect pausing at a downstream nu-
cleosome , similar to pol II accumulation downstream of its tran-
scribed genes . Accordingly, it seems possible that nucleosomes
abutting terminators could increase pausing and thereby modulate
termination and/or pol III release and reinitiation.
7. Coupling of pol III termination with RNA processing
As noted above, data indicate that the C11-mediated RNA 3′ cleav-
age that occurs during termination is responsible, at least in part, for
the variable lengths of the oligo(U) termini of several nascent pol III
transcripts [recently reviewed in 77]. The 1–2 nucleotide increase in
3′ oligo(U) length can have striking effects on pre-tRNA turnover
and maturation in vivo . The subtle increase in 3′ oligo(U) length
that occurs in C11 cleavage mutants significantly increases the affini-
ty of the transcripts for La protein with consequent protection from
transcript degradation, more efficient RNA processing and increased
levels of mature functional tRNA . In the absence of La binding
pre-tRNAs may succumb to nuclear surveillance-mediated decay by
the exosome . Indeed La levels are limiting for tRNA maturation
in S. pombe, but increasing La by ectopic expression or increasing the
3′ oligo(U) length on pre-tRNAs in C11 cleavage mutants drives
more nascent transcripts into the La-dependent productive path-
way of tRNA maturation . These observations suggest a role
for termination-associated C11-mediated transcript cleavage in af-
fecting functional tRNA production and raises the possibility that
termination-associated nibbling by C11 helps control tRNA levels
and thus translation. Consistent with this possibility is that a
genome-wide search for candidate factors regulated by upstream
open reading frames (uORFs) similar to uORFs that regulate GCN4
under general translational stress revealed C11 as one of just a
few S. cerevisiae genes .
While La has been found to transiently bind to all of the different
types of nascent pol III transcripts examined, and its effects are
most extensively characterized for tRNAs [reviewed in 77], it is also
functionally involved in U6 snRNA maturation [158,159]. As detailed
below this may reflect a conserved mechanistic coupling between
pol III transcription termination and U6 RNA 3′ processing which
involves an intricate series of what appear to be 3′ uridylate-specific
activities. Mature U6 snRNA has a 2′–3′ cyclic phospho-UMP residue
at its 3′ end, formed as part of a deuridylation–urydylation reac-
tion . The sequential terminal Us on pre-U6 snRNA that are
A.G. Arimbasseri et al. / Biochimica et Biophysica Acta 1829 (2013) 318–330
formed as a result of pol III termination may be required for this pro-
cess. La is a functional component of U6 3′ end metabolism [158,159].
Curiously, U6 snRNA is transcribed in all species by pol III, while
the other spliceosomal RNAs (U1, U2, U4, U5) are transcribed by pol
II. However, unlike tRNA and 5S, the U6 snRNA sequence apparently
cannot accommodate an internal B box promoter element. Different
species have developed varying mechanisms to circumvent the prob-
lematic need for a B box sequence in functional U6 snRNA genes. S.
cerevisiae has the B box downstream of the terminator ; binding
of TFIIIC to the A and B box elements is facilitated by a positioned nu-
cleosome between them [147,149]. An alternative solution was
achieved by S. pombe, in which the B box resides in an intron in the
U6 gene that is spliced out upon maturation of U6 snRNA . Ver-
tebrates use yet another strategy: entirely upstream promoters for
pol III-mediated U6 transcription . In vertebrates this additional-
ly involved the emergence of a Brf1-homologous protein known as
Brf2 . These observations suggest that U6 snRNA production
can accommodate a variety of promoter types and positions which
in some cases employ specially positioned nucleosomes, intron inser-
tion, and factor-specific modes of initiation by pol III. These examples
suggest that it is important that the U6 snRNA is a pol III transcript in
these species and that the intricate uridylate-specific 3′ end process-
ing accounts for its dependence on pol III.
The coupling of a pol III termination mechanism that produces
RNAs with 3′ oligo(U) ends that mediate specific association with
the La protein maturation factor is undoubtedly a means to afford
pol III transcripts a processing pathway separate from the pol I and
II transcript maturation pathways.
It was recently reported that pol III transcription and precursor
tRNA processing are linked via a pathway that involves the pol III re-
pressor Maf1, although this is likely due to saturation of processing
and/or export machinery . A role for RNAse P in coupling pol
III transcription and tRNA 5′ processing has been proposed
[164,165], whereas all other links between the transcription and
processing of class III genes appear to be via 3′ end processing as de-
8. Concluding remarks
Transcription termination involves pausing followed by destabili-
zation of the elongation complex with dissociation of its components.
Bacterial RNA polymerase as well as pol II can terminate transcription
in response to more than one terminal signal. Pol II termination of
poly(A)-containing mRNA synthesis uses a cis-acting terminator ele-
ment, the poly(A) addition site, that acts at a distance to initiate the
process, followed by dissociation of the complex somewhere down-
stream, somewhat similar to the Rho-dependent mechanism of
E. coli transcription termination. Pol III can achieve the same outcome
on a short oligo(dA) template that serves to both pause and dissociate
the complex with efficiency and near nucleotide precision, somewhat
similar to the intrinsic termination by E. coli RNA polymerase. Yet, de-
spite the efficiency of what appears to be a simple termination signal
for pol III, involvement of multiple subunits suggests a complex
mechanism of termination. Recent reports of termination defective
mutants of C37 and C11, and localization of these plus C53 near the
catalytic site suggest active alteration of the pol III active center dur-
ing termination, rather than passive cessation of RNA synthesis. Con-
trol of elongation rate as well as the degree to which the oligo(rU:dA)
hybrid confers instability to the pol III complex are likely underlying
components of the termination mechanism that are sensitive to the
activities of the termination subunits. Because C37, C53 and C11
have homologs in the pol I and II systems, it is suggested that these
polymerases may use similar mechanisms to execute their final
stages of termination, pausing, complex destabilization, dissociation
High-resolution crystal structures of pol III elongation complexes
with and without C11/C53 and C11 will undoubtedly reveal more
about these subunits and their involvement in the catalytic center.
Another challenge will be to obtain crystals of a pol III termination
An aspect that we believe will provide unique insight into the
mechanisms of pol III termination is to understand the species-
specific differences in the minimal number of Ts required, as well as
oligo(dT) context-dependent effects, and how terminator-adjacent
binding sites for DNA-binding factors may promote termination. In ad-
dition, ‘secondary terminators’ and non-canonical pol III terminators
are also a potential trove of important means to production of a
newly emerging class of tRNA-downstream short RNAs.
This work was supported by the Intramural Research Program of
the Eunice Kennedy Shriver National Institute of Child Health and
Human Development, NIH.
 F. Werner, D. Grohmann, Evolution of multisubunit RNA polymerases in the
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