16.5–19.0 h (short-period mutants) to 24.0–
29.0 h (long-period mutants) or to be
completely arrhythmic (Feldman and Hoyle
1973; Gardner and Feldman 1980; Aronson
et al. 1994). Many of the mutations in these
short- and long-period strains mapped to a
single locus, which was dubbed frequency (frq)
(Feldman and Hoyle 1973; Gardner and Feldman 1980, 1981; Loros et al. 1986).
Full-length FRQ is a largely disordered 989
aa long protein containing several smaller
potentially well-ordered domains that are
involved in the formation of the negative arm
protein complex. Due to the low proportion of
in silico predicted structure, FRQ was believed
to be an intrinsically disordered protein (IDP),
a protein that lacks a fixed or ordered threedimensional structure instead sampling a
hetergenous ensemble of conformations. This
prediction of disorder was demonstrated biochemically as FRQ remained soluble after heat
treatment and was more quickly digested by
proteases (Hurley et al. 2013). While FRQ does
not have sequence homologs amongst all higher
eukaryotes, intrinsic disorder does appear to be
conserved among negative arm proteins, suggesting that disorder is a key feature of negative
arm proteins (Hurley et al. 2013).
There are two isoforms of FRQ that are the products of
alternate translation start sites, Long-FRQ (989 aa) and
Short-FRQ (889 aa, missing the first 100 amino acids),
which are thought to contribute to temperature compensation of the clock (Garceau et al. 1997), as previously discussed in Sect. II.B. Both FRQ isoforms
encompass several key protein-protein interaction
domains, including the coiled-coil interaction domain
that allows for the dimerization of FRQ (necessary for
rhythmicity; Cheng et al. 2001a), the nuclear localization region (Luo et al. 1998), and the FRQ-CK1 interacting domains FCD1 and FCD2 (Querfurth et al. 2011).
In addition, both isoforms contain two predicted proline, glutamic acid, serine, threonine (PEST) domains
that may be important for protein turnover, PEST-1
and PEST-2 (Merrow and Dunlap 1994). Thus far, the
only in vivo evidence that these domains act in protein
turnover is that the PEST-1 region is phosphorylated
before FRQ is degraded (Liu et al. 2000; Go ¨rl et al. 2001;
Baker et al. 2009). Finally, the isoforms include an FRQFRH interacting domain called the FFD (Guo et al.
2010). These interaction domains contribute to the
formation of the larger negative arm complex, the
Frequency-FRH Complex (FFC) (Baker et al. 2009;
Guo et al. 2010; Querfurth et al. 2011; Hurley et al.
2013).
FRQ transcript and protein levels oscillate
on a daily basis as a result of the activity of the
WCC. At the beginning of the circadian cycle,
WCC activates frq expression, and approximately 4 h later there is a peak in translated
FRQ levels (Garceau et al. 1997; Merrow et al.
1997). Shortly after translation, FRQ dimerizes
with itself and binds to FRQ-interacting RNA
Helicase (FRH) to form the FRQ/FRH complex
(FFC); all FRQ is found in complex with FRH in
a two to one ratio (Cheng et al. 2001a, 2005;
Baker et al. 2009). This interaction helps to
stabilize FRQ and is discussed further in Sect.
III.B.2 (Cheng et al. 2005; Hurley et al. 2013).
Many kinases act to phosphorylate FRQ (see
Sect. III.D for more details) and this phosphorylation allows the FFC to enter the nucleus early
on in the cycle to repress WCC activation of frq
transcription, closing the negative feedback
loop (Fig. 4.2) (Cheng et al. 2005; Hong et al.
2008; Baker et al. 2009; Cha et al. 2011). Phosphorylation eventually leads to FRQ degradation via the ubiquitin-proteasome pathway
(Fig. 4.2), and a new wave of FRQ protein is
translated as the circadian cycle continues (He
and Liu 2005a; Larrondo et al. 2015).
In addition to direct WCC regulation, further transcriptional regulation supports robust
oscillations in frq mRNA levels. The WCC activates transcription of frq’s long non-coding
antisense transcript, qrf (frq spelled backwards) in response to light by utilizing a promoter in the 3
0 UTR of frq, and this aids in light
resetting of the clock (Kramer et al. 2003). In
constant darkness, qrf expression is also regulated by another unknown mechanism (Xue
et al. 2014). Data suggests that qrf silences frq
expression to tune the timing and period of the
clock, yielding more robust oscillations of frq
by counteracting any “leaky” frq expression
that would lead to dampening of the central
oscillator. However, two competing models
suggest two distinct mechanisms as to how
this occurs. These include either the stalling/
collision of the RNA polymerases transcribing
86
M. S. Jankowski et al.
29.0 h (long-period mutants) or to be
completely arrhythmic (Feldman and Hoyle
1973; Gardner and Feldman 1980; Aronson
et al. 1994). Many of the mutations in these
short- and long-period strains mapped to a
single locus, which was dubbed frequency (frq)
(Feldman and Hoyle 1973; Gardner and Feldman 1980, 1981; Loros et al. 1986).
Full-length FRQ is a largely disordered 989
aa long protein containing several smaller
potentially well-ordered domains that are
involved in the formation of the negative arm
protein complex. Due to the low proportion of
in silico predicted structure, FRQ was believed
to be an intrinsically disordered protein (IDP),
a protein that lacks a fixed or ordered threedimensional structure instead sampling a
hetergenous ensemble of conformations. This
prediction of disorder was demonstrated biochemically as FRQ remained soluble after heat
treatment and was more quickly digested by
proteases (Hurley et al. 2013). While FRQ does
not have sequence homologs amongst all higher
eukaryotes, intrinsic disorder does appear to be
conserved among negative arm proteins, suggesting that disorder is a key feature of negative
arm proteins (Hurley et al. 2013).
There are two isoforms of FRQ that are the products of
alternate translation start sites, Long-FRQ (989 aa) and
Short-FRQ (889 aa, missing the first 100 amino acids),
which are thought to contribute to temperature compensation of the clock (Garceau et al. 1997), as previously discussed in Sect. II.B. Both FRQ isoforms
encompass several key protein-protein interaction
domains, including the coiled-coil interaction domain
that allows for the dimerization of FRQ (necessary for
rhythmicity; Cheng et al. 2001a), the nuclear localization region (Luo et al. 1998), and the FRQ-CK1 interacting domains FCD1 and FCD2 (Querfurth et al. 2011).
In addition, both isoforms contain two predicted proline, glutamic acid, serine, threonine (PEST) domains
that may be important for protein turnover, PEST-1
and PEST-2 (Merrow and Dunlap 1994). Thus far, the
only in vivo evidence that these domains act in protein
turnover is that the PEST-1 region is phosphorylated
before FRQ is degraded (Liu et al. 2000; Go ¨rl et al. 2001;
Baker et al. 2009). Finally, the isoforms include an FRQFRH interacting domain called the FFD (Guo et al.
2010). These interaction domains contribute to the
formation of the larger negative arm complex, the
Frequency-FRH Complex (FFC) (Baker et al. 2009;
Guo et al. 2010; Querfurth et al. 2011; Hurley et al.
2013).
FRQ transcript and protein levels oscillate
on a daily basis as a result of the activity of the
WCC. At the beginning of the circadian cycle,
WCC activates frq expression, and approximately 4 h later there is a peak in translated
FRQ levels (Garceau et al. 1997; Merrow et al.
1997). Shortly after translation, FRQ dimerizes
with itself and binds to FRQ-interacting RNA
Helicase (FRH) to form the FRQ/FRH complex
(FFC); all FRQ is found in complex with FRH in
a two to one ratio (Cheng et al. 2001a, 2005;
Baker et al. 2009). This interaction helps to
stabilize FRQ and is discussed further in Sect.
III.B.2 (Cheng et al. 2005; Hurley et al. 2013).
Many kinases act to phosphorylate FRQ (see
Sect. III.D for more details) and this phosphorylation allows the FFC to enter the nucleus early
on in the cycle to repress WCC activation of frq
transcription, closing the negative feedback
loop (Fig. 4.2) (Cheng et al. 2005; Hong et al.
2008; Baker et al. 2009; Cha et al. 2011). Phosphorylation eventually leads to FRQ degradation via the ubiquitin-proteasome pathway
(Fig. 4.2), and a new wave of FRQ protein is
translated as the circadian cycle continues (He
and Liu 2005a; Larrondo et al. 2015).
In addition to direct WCC regulation, further transcriptional regulation supports robust
oscillations in frq mRNA levels. The WCC activates transcription of frq’s long non-coding
antisense transcript, qrf (frq spelled backwards) in response to light by utilizing a promoter in the 3
0 UTR of frq, and this aids in light
resetting of the clock (Kramer et al. 2003). In
constant darkness, qrf expression is also regulated by another unknown mechanism (Xue
et al. 2014). Data suggests that qrf silences frq
expression to tune the timing and period of the
clock, yielding more robust oscillations of frq
by counteracting any “leaky” frq expression
that would lead to dampening of the central
oscillator. However, two competing models
suggest two distinct mechanisms as to how
this occurs. These include either the stalling/
collision of the RNA polymerases transcribing
86
M. S. Jankowski et al.