S. cerevisiae DNA
binding protein RAP-1
Mattias Galliano '15 and Tomas Grant '16
The Saccharomyces cerevisiae Ras-proximate-1 or
Ras-related protein 1 (RAP1) is a DNA binding protein involved with
transcriptional regulation through binding of telomeric DNA. The
protein contains two similar domains that bind the DNA in a tandem
orientation, that recognizes a tandemly repeated DNA sequence.
Telomeres are dynamic structures and recent observations indicate that
the shortening of telomeres may be involved in both aging and cancer.
The best characterized telomeres, both in terms of structure and
function, are those of the budding yeast S. cerevisiae, and
has been shown to be bound by the abundant, non-histone protein RAP1.
RAP-1 is considered to be the major sequence-specific
DNA-binding protein in yeast telomeres and is directly responsible
for regulating telomere length. It has been shown that RAP1 binding
sites are found in promoters of a large number of genes, including
ribosomal protein genes. RAP1 also functions as a transcriptional
activator and repressor, and is responsible for recruiting the
transcription factors SIR3/SIR4, which gives rise to transcriptional
silencing and heterochromatin formation.
II. General Structure
RAP1 is a large protein of 827 amino acid residues that
contain distinct functional domains. The minimal DNA-binding domain
(RAP1 DBD) resides in the middle of the protein between residues 361
and 596, an unusually large domain with 236 residues and as a whole
is completely unhomologous to any DNA-binding motifs known to date.
The overall structure of RAP1 DBD consists of two clearly defined
domains;the N-terminal domain (DOMAIN
I) and the C-terminal domain (DOMAIN
II). The complex shown here consists of two nearly identical
complexes in an asymmetric unti aligned head-to-head, with the DNA
forming pseudo-continuous fibers in the crystal strucure.
Each domain contains a three-helix bundle and an
N-terminal arm, which make specific contacts with bases in the major
and minor groove.
The N-terminal domain is
responsible for dimerization and DNA
binding, and is also responsible for DNA bending. The RAP1 DBD
consists of two domains which are connected by a linker
which crosses the minor groove between two ACACC repeats
containing both the sugar-phosphate backbone and a base in the
minor groove. Overall RAP1 binds as a dimer with an affinity for
5'-ACACCCACACACC-3', and one RAP1 molecule binds every 18 bp of
DNA and RAP1 binds essentially continuously along the double
strandered telomeric DNA.
III. Domain I
Domain 1 consists of a short N-terminal arm (360-364), a
three-helix bundle (H1A: 365-376, H1B:386-391, H1C: 400-409), and
a region of loosely folded structure (410-445)
Direct side-chain interactions between the protein and the
DNA it's binding are hydrogen bonds occuring at Asn
401, , and and
Arg-404 to G8'-G9'.
H1C serves as the DNA recognition helix
within domain 1 of the RAP1 DBD,and along with H1B and the
N-terminal arm constitute a helix-turn-helix
DNA binding motif, responsible for initial DNA
recognition, and is homologous to known DNA-binding motifs.
Specific water mediated
interactions involve three well positioned water molecules
leads to sequence-specific contacts to C6 and G8 through
His-405, Arg 408, , and
Arg-404 as well.
BUTTON Hydrophobic interaction (
Ser 402 and His-405 with C6) increases specificity of
IV. DOMAIN II
Once RAP1 DBD I has bound DNA, Domain II is now
prepared to bind to the DNA.
In this protein-DNA model we can see two repeating sequences
of DNA bound by two seperate RAP1 proteins seperated by
approximately 18 bp. Binding occurs at the conserved
sequence of 5'-ACACCCACACACC-3'.
Domain II is similar in structure to Domain I
with a three-helix bundle (H2A: 451-470, H2B: 525-532,
H2C:538-552) and a N-terminal arm (residues 446-450).
Domain I differs from RAP1 D1 in the fact that it posesses
a fourth helix structure H2D, which interacts with H2A to
stabilize the DNA-protein interactions occuring between
the DNA binding site on RAP1 and the DNA being contacted.
Domain II contains a similar
helix to that found in Domain I, and has a
similar orientation but makes distinctly differet
contacts to the DNA phosphate backbone and its bases.
The DNA-recognition helix H2C is longer than the
corrseponding helix H1C of domain 1 and contains an
alpha-helical bulge, which results from the insertion of
an additional residue of Phe-548 into a helical turn.
of minor groove by N-terminal arm(446-450) of domain 2 with Ser-444
and Ile-445 at the C-terminus of domain 1)
between Domain II and DNA bases involve A13-C14-A15-C16-C17 of the
3'region of DNA-binding site and Arg-542, Asp-543,
Arg-546, Lys-547 all from recognition helix H2C.
Konig, et al. 1996. The Crystal
Structure of the DNA-Binding Domain of Yeast RAP1 in Complex
with Telomeric DNA.The Cell. 85: 125-136.
Johan Wahlin and Marita Cohn. 2000.
Saccharomyces cerevisiae RAP1 binds to telomeric
sequences with spatial flexibility. Nucleic Acids
Research 28(12): 2292-2301.
Jason D. Lieb, Xiaole Liu, David Botstein, and
Patrick O. Brown. 2001. Promoter-specific binding of Rap1
revealed by genome-wide maps of protein-DNA association. Nature
A.L. Henry Yves et al. 1990.
Characterization of the DNA binding domain of the yeast
Rap1 protein Nucleic Acids Research
Martinez, et al. 2010. Mammalian
Rap1 controls telomere function and gene expression
through binding to telomeric and extratelomeric sites. Nature
Cell Biology 12: 768-780.
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