S. cerevisiae DNA binding protein RAP-1

Mattias Galliano '15 and Tomas Grant '16


I. Introduction

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 interactions with His-405, Arg 408, , and Arg-404 as well.

BUTTON Hydrophobic interaction ( Ser 402 and His-405 with C6) increases specificity of recognition


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 recognition 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)

Specific 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.

V. References

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 Genetics 28:327-334.

A.L. Henry Yves et al. 1990. Characterization of the DNA binding domain of the yeast Rap1 protein Nucleic Acids Research 18(9):2617-2623.

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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