The carboxy‐terminal domain of the LexA repressor oligomerises essentially as the entire protein

Heterochromatin is nucleated at a specific site and subsequently spreads into distal sequences through multiple interactions between modified histones and nonhistone proteins. In the yeast Saccharomyces cerevisiae, these nonhistone proteins include Sir2, Sir3, and Sir4. We have previously shown that loss of the C-terminal Rap1 domain containing Sir3 and Sir4 association sites can be overcome by tethering a 144-amino-acid C-terminal domain (CTD) of Sir3 adjacent to the telomere. Here, we explore the substructure and functions of the CTD. We demonstrate that the CTD is the minimum domain for Sir3 homodimerization, a function that is conserved in related yeasts. However, CTD heterodimers associate at only low efficiencies and correspondingly have low levels of tethered silencing, consistent with an essential role for dimerization in tethered silencing. Six missense alleles were generated, with ctd-Y964A producing the most extreme phenotypes when tethered to the LexA binding sites. Although ctd-Y964A is capable of dimerization, telomere silencing is abrogated, indicating that the CTD serves a second essential function in silencing. Chromatin immunoprecipitation analyses of wild-type and ctd-Y964A mutant cells indicate an association of the CTD with the deacetylated histone tails of H3 and H4 that is necessary for the recruitment of Sir3. The efficiency of spreading depends upon the apparent stoichiometry and stability during the initiation event. The predicted Cdc6 domain III winged-helix structure may well be responsible for dimerization.

Section: Downloaded Fromsupporting

confidence: 77%

Sir3 C-Terminal Domain Involvement in the Initiation and Spreading of Heterochromatin

Liaw

Lustig

2006

“…This LexA segment does not contain nuclear localization signals or the DNA dimerization region thought to be required for stable interaction with the LexA operator (22,51). By comparison, we were able to identify and define independent activation sequences when Act domains were fused instead to the DNAbinding domain of yeast activator GAL4, which provides localization and dimerization functions (unpublished data).…”

Section: Resultsmentioning

confidence: 91%

Homotypic Interactions of Chicken GATA-1 Can Mediate Transcriptional Activation

Yang

Evans

1995

We used a one-hybrid system to replace precisely the finger II chicken GATA-1 DNA-binding domain with the binding domain of bacterial repressor protein LexA. The LexA DNA-binding domain lacks amino acids that function for transcriptional activation, nuclear localization, or protein dimerization. This allowed us to analyze activities of GATA-1 sequences distinct from DNA binding. We found that strong transcriptional activating sequences that function independently of finger II are present in GATA-1. Sequences including finger I contain an independent nuclear localizing function. Our data are consistent with cooperative binding of two LexA-GATA-1 hybrid proteins on a palindromic operator. The sensitivity of our transcription assay provides the first evidence that GATA-1 can make homotypic interactions in vivo. The ability of a non-DNA-binding form of GATA-1 to activate gene expression by targeting to a bound GATA-1 derivative further supports the notion that GATA-1-GATA-1 interactions may have functional consequences. A coimmunoprecipitation assay was used to demonstrate that GATA-1 multimeric complexes form in solution by protein-protein interaction. The novel ability of GATA-1 to interact homotypically may be important for the formation of higher-order structures among distant regulatory elements that share binding sites for this transcription factor. We also used the system to test the ability of GATA-1 to interact heterotypically with other activators.

“…6A suggests that LexA-GCN2 forms homodimers with itself more efficiently than it forms heterodimers with native GCN2. This preference may reflect the fact that LexA itself has a dimerization function (27). The formation of homodimers may also be favored if dimerization is coincident with translation.…”

Section: Resultsmentioning

confidence: 99%

Dimerization by Translation Initiation Factor 2 Kinase GCN2 Is Mediated by Interactions in the C-Terminal Ribosome-Binding Region and the Protein Kinase Domain

Qiu

Garcia-Barrio

Hinnebusch

1998

The protein kinase GCN2 stimulates translation of the transcriptional activator GCN4 in yeast cells starved for amino acids by phosphorylating translation initiation factor 2. Several regulatory domains, including a pseudokinase domain, a histidyl-tRNA synthetase (HisRS)-related region, and a C-terminal (C-term) segment required for ribosome association, have been identified in GCN2. We used the yeast two-hybrid assay, coimmunoprecipitation analysis, and in vitro binding assays to investigate physical interactions between the different functional domains of GCN2. A segment containing about two thirds of the protein kinase (PK) catalytic domain and another containing the C-term region of GCN2 interacted with themselves in the two-hybrid assay, and both the PK and the C-term domains could be coimmunoprecipitated with wild-type GCN2 from yeast cell extracts. In addition, in vitro-translated PK and C-term segments showed specific binding in vitro to recombinant glutathione S-transferase (GST)-PK and GST-C-term fusion proteins, respectively. Wild-type GCN2 could be coimmunoprecipitated with a full-length LexA-GCN2 fusion protein from cell extracts, providing direct evidence for dimerization by full-length GCN2 molecules. Deleting the C-term or PK segments abolished or reduced, respectively, the yield of GCN2-LexA-GCN2 complexes. These results provide in vivo and in vitro evidence that GCN2 dimerizes through self-interactions involving the C-term and PK domains. The PK domain showed pairwise in vitro binding interactions with the pseudokinase, HisRS, and C-term domains; additionally, the HisRS domain interacted with the C-term region. We propose that physical interactions between the PK domain and its flanking regulatory regions and dimerization through the PK and C-term domains both play important roles in restricting GCN2 kinase activity to amino acid-starved cells.