ISSN 1022-7954, Russian Journal of Genetics, 2008, Vol. 44, No. 2, pp. 131–136. © Pleiades Publishing, Inc., 2008.
Original Russian Text © D.V. Kopytova, Yu.V. Nikolenko, L.A. Lebedeva, E.N. Nabirochkina, Yu.V. Shidlovskii, S.G. Georgieva, A.N. Krasnov, 2008, published in Genetika, 2008,
Vol. 44, No. 2, pp. 163–169.
To initiate transcription, each RNA polymerase
needs a certain set of general transcription factors. The
TFIID complex, consisting of the TATA box-binding
protein (TBP) and TBP-associated factors (TAF), rec-
ognizes speciﬁc protein sequences. The C-terminal
domain of TBP is highly conserved among eukaryotes
and harbors two symmetrical repeats, which form a
saddle-like structure essential for the interaction with a
promoter sequence [1, 2].
Metazoa have a second gene for a TBP-like factor
(TLF, also known as TRF2 or TLP), which is highly
homologous to the main TBP domain (approximately
39% homology) [3, 4]. This DNA-binding domain is
highly conserved among all TBP-related proteins. The
residues responsible for the binding of general tran-
scription factors TFIIA and TFIIB have 60 and 75%
homology, respectively. However, the residues essential
for the interaction of TBP with the TATA box are
replaced by other residues in TRF2, and, consequently,
recombinant TRF2 does not bind the canonical TATA
box [3–6]. TLF mediates transcription initiation by
RNA polymerase II and interacts with TFIIA and
TFIIB during the formation of the preinitiation com-
plex. However, TLF does not bind to classical TATA
sequences and controls transcription of a set of genes
that are not controlled by TBP [6–8].
Analysis of the polytene chromosomes has shown that
TLF, termed TBP-related factor 2 (TRF2),
occurs in sites other than the sites of TBP localization. In
addition, TRF2 is a component of a 500-kDa complex
[3, 6, 8], which includes components of the NURF
chromatin-remodeling complex and the DRE (DNA
replication-related element) binding factor (DREF).
The TRF2–DREF complex activates transcription of
the genes whose promoter harbors DRE [9–11].
The objective of this work was to study the structure
gene and the properties of its protein product.
MATERIALS AND METHODS
Cloning of the trf2 cDNA.
To map the transcripts
corresponding to the region of interest, we ampliﬁed
the 5' and 3' cDNA ends by RACE. Analysis of the
RACE products showed that the 5' end of mRNA is
1.2 kb upstream of the
-element insertion, while the 3'
end is 1 kb downstream of the end of the open reading
frame (ORF). We designed gene-speciﬁc primers,
which were then used to synthesize the full-length cDNA.
The 5'- and 3'-RACE reactions and PCR were carried out
with a cDNA ampliﬁcation kit (Clontech) and primers
AGGATCGTAAACGGGTATAT (5'-RACE), GCG-
GCTAGCGTAAACCACGTTG (3'-RACE), GTATCGT-
GTGTGCTCAGACTTGTGCTCTCA, and GCAGCGC-
TAGCTTAGAAGGGCATATCCA (ampliﬁcation of the
RNA isolation and Northern blotting.
RNA was isolated from
vae, pupae, and adults with Trizol (Invitrogen) as recom-
mended by the manufacturer. Poly(A)
RNA was puriﬁed
on an oligo(dT)-cellulose column. A poly(A)
ple (1.5 mg) was applied onto agarose gel and transferred
onto a nitrocellulose membrane. Hybridization products
were visualized using a Cyclone Storage Phosphor Sys-
tem (Packard Instrument Company). Probes correspond-
ing to various ORF regions and used for Northern blot-
ting were PCR-ampliﬁed with two pairs of primers:
TCAAGTTAATCGCGAGTACC and AGGATCG-
TAAACGGGTATAT in the case of probe 1 and TAAG-
Study of the
Drosophila melanogaster trf2
and Its Protein Product
D. V. Kopytova
, Yu. V. Nikolenko
, L. A. Lebedeva
, E. N. Nabirochkina
Yu. V. Shidlovskii
, S. G. Georgieva
a, b, c
, and A. N. Krasnov
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334 Russia; e-mail: firstname.lastname@example.org
University of Oslo, Center of Medical Research in Russia, Moscow, 119334 Russia
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 117984 Russia
Received April 19, 2007
TRF2 protein regulates transcription of several genes. The
structure was studied. The gene proved to code for two protein isoforms, a known 75-kDa isoform and a newly
identiﬁed 175-kDa isoform. The new isoform combines the known isoform sequence with an extended N end
containing a coiled-coil motif. The long TRF2 isoform was found to act as a component of a multiprotein com-
plex, including ISWI ATPase as well.