scholarly journals Processivity of the Saccharomyces cerevisiae Poly(A) Polymerase Requires Interactions at the Carboxyl-Terminal RNA Binding Domain

1998 ◽  
Vol 18 (10) ◽  
pp. 5942-5951 ◽  
Author(s):  
Alexander Zhelkovsky ◽  
Steffen Helmling ◽  
Claire Moore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Rna Binding ◽  
Atp Binding Site ◽  
Nucleotide Binding Sites ◽  
Carboxyl Terminal ◽  
Unique Specificity ◽  
Carboxy Terminal

ABSTRACT The interaction of the Fip1 subunit of polyadenylation factor I with the Saccharomyces cerevisiae poly(A) polymerase (PAP) was assayed in vivo by two-hybrid analysis and was found to involve two separate regions on PAP, located at opposite ends of the protein sequence. In vitro, Fip1 blocks access of the RNA primer to an RNA binding site (RBS) that overlaps the Fip1 carboxy-terminal interaction region and, in doing so, shifts PAP to a distributive mode of action. Partial truncation of this RBS has the same effect, indicating that this site is required for processivity. A comparison of the utilization of ribo- and deoxyribonucleotides as substrates indicates the existence on PAP of a second RBS which recognizes the last three nucleotides at the 3′ end of the primer. This site discriminates against deoxyribonucleotides at the 3′ end, and interactions at this site are not affected by Fip1. Further analysis revealed that the specificity of PAP for adenosine is not simply a function of the ATP binding site but also reflects interactions with bases at the 3′ end of the primer and at another contact site 14 nucleotides upstream of the 3′ end. These results suggest that the unique specificity of PAP for ribose and base, and thus the extent and type of activity with different substrates, depends on interactions at multiple nucleotide binding sites.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

Ligand-induced phosphorylation of the colony-stimulating factor 1 receptor can occur through an intermolecular reaction that triggers receptor down modulation

1990 ◽  
Vol 10 (4) ◽  
pp. 1664-1671
Author(s):  
M Ohtsuka ◽  
M F Roussel ◽  
C J Sherr ◽  
J R Downing
Keyword(s):  
Colony Stimulating Factor ◽  
Intact Cells ◽  
Atp Binding Site ◽  
Truncation Mutant ◽  
Intermolecular Mechanism ◽  
Carboxy Terminal ◽  
Lysine Substitution ◽  
Stimulating Factor

Ligand-induced tyrosine phosphorylation of the human colony-stimulating factor 1 receptor (CSF-1R) could involve either an intra- or intermolecular mechanism. We therefore examined the ability of a CSF-1R carboxy-terminal truncation mutant to phosphorylate a kinase-defective receptor, CSF-1R[met 616], that contains a methionine-for-lysine substitution at its ATP-binding site. By using an antipeptide serum that specifically reacts with epitopes deleted from the enzymatically competent truncation mutant, cross-phosphorylation of CSF-1R[met 616] on tyrosine was demonstrated, both in immune-complex kinase reactions and in intact cells stimulated with CSF-1. Both in vitro and in vivo, CSF-1R[met 616] was phosphorylated on tryptic peptides identical to those derived from wild-type CSF-1R, suggesting that receptor phosphorylation on tyrosine can proceed via an intermolecular interaction between receptor monomers. When expressed alone, CSF-1R[met 616] did not undergo ligand-induced down modulation, but its phosphorylation in cells coexpressing the kinase-active truncation mutant accelerated its degradation.

scholarly journals A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

scholarly journals Ligand-induced phosphorylation of the colony-stimulating factor 1 receptor can occur through an intermolecular reaction that triggers receptor down modulation.

1990 ◽  
Vol 10 (4) ◽  
pp. 1664-1671 ◽  
Author(s):  
M Ohtsuka ◽  
M F Roussel ◽  
C J Sherr ◽  
J R Downing
Keyword(s):  
Colony Stimulating Factor ◽  
Intact Cells ◽  
Atp Binding Site ◽  
Truncation Mutant ◽  
Intermolecular Mechanism ◽  
Carboxy Terminal ◽  
Lysine Substitution ◽  
Stimulating Factor

Ligand-induced tyrosine phosphorylation of the human colony-stimulating factor 1 receptor (CSF-1R) could involve either an intra- or intermolecular mechanism. We therefore examined the ability of a CSF-1R carboxy-terminal truncation mutant to phosphorylate a kinase-defective receptor, CSF-1R[met 616], that contains a methionine-for-lysine substitution at its ATP-binding site. By using an antipeptide serum that specifically reacts with epitopes deleted from the enzymatically competent truncation mutant, cross-phosphorylation of CSF-1R[met 616] on tyrosine was demonstrated, both in immune-complex kinase reactions and in intact cells stimulated with CSF-1. Both in vitro and in vivo, CSF-1R[met 616] was phosphorylated on tryptic peptides identical to those derived from wild-type CSF-1R, suggesting that receptor phosphorylation on tyrosine can proceed via an intermolecular interaction between receptor monomers. When expressed alone, CSF-1R[met 616] did not undergo ligand-induced down modulation, but its phosphorylation in cells coexpressing the kinase-active truncation mutant accelerated its degradation.

scholarly journals A role for CDC7 in repression of transcription at the silent mating-type locus HMR in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (2) ◽  
pp. 1080-1091 ◽  
Author(s):  
A Axelrod ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Mating Type ◽  
Double Mutant ◽  
Temperature Sensitive ◽  
Alpha Cells ◽  
Nucleotide Substitutions ◽  
Type Locus

The mating-type genes at MAT in Saccharomyces cerevisiae are expressed, whereas the same genes located at HML and HMR are transcriptionally repressed. The DNA element responsible for repression at HMR has been termed a silencer and contains an autonomous replication sequence, a binding site for GRFI/RAPI, and a binding site for ABFI. A double-mutant HMR-E silencer that contains single nucleotide substitutions in both the GRFI/RAPI- and ABFI-binding sites no longer binds either factor in vitro, nor represses transcription at HMR in vivo. In MAT alpha cells, this derepression of a information results in a nonmating phenotype. Second-site suppressor mutations were isolated that restored the alpha mating phenotype to MAT alpha cells containing the double-mutant silencer. One of these suppressors, designated sas1-1, conferred a temperature-sensitive lethal phenotype to the cell. SAS1 was found to be identical to CDC7, a gene which encodes a protein kinase required for the initiation of DNA replication. This new allele of CDC7 was designated cdc7-90. cdc7-90 restored the alpha mating phenotype by restoring silencing. The original allele of CDC7, isolated on the basis of the cell cycle phenotype it confers, also restored silencing, and overexpression of CDC7 interfered with silencing. cdc7-90 did not restore detectable binding of GRFI/RAPI or ABFI to the double-mutant silencer in vitro. These results indicate that a reduced level of CDC7 function restores silencing to a locus defective in binding two factors normally required for silencing.

scholarly journals The Yeast hnRNP-Like Proteins Yra1p and Yra2p Participate in mRNA Export through Interaction with Mex67p

2001 ◽  
Vol 21 (13) ◽  
pp. 4219-4232 ◽  
Author(s):  
Daniel Zenklusen ◽  
Patrizia Vinciguerra ◽  
Yvan Strahm ◽  
Françoise Stutz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Protein ◽  
Rna Binding ◽  
Mrna Export ◽  
Deletion Mutants ◽  
Variable Regions ◽  
Evolutionarily Conserved ◽  
Rna Export

ABSTRACT Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessentialSaccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)+ RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

scholarly journals Role for Upf2p Phosphorylation in Saccharomyces cerevisiae Nonsense-Mediated mRNA Decay

2006 ◽  
Vol 26 (9) ◽  
pp. 3390-3400 ◽  
Author(s):  
Weirong Wang ◽  
Iván J. Cajigas ◽  
Stuart W. Peltz ◽  
Miles F. Wilkinson ◽  
Carlos I. González
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Nonsense Mediated Mrna Decay ◽  
Higher Eukaryotes ◽  
Nonsense Codons ◽  
First Time

ABSTRACT Premature termination (nonsense) codons trigger rapid mRNA decay by the nonsense-mediated mRNA decay (NMD) pathway. Two conserved proteins essential for NMD, UPF1 and UPF2, are phosphorylated in higher eukaryotes. The phosphorylation and dephosphorylation of UPF1 appear to be crucial for NMD, as blockade of either event in Caenorhabditis elegans and mammals largely prevents NMD. The universality of this phosphorylation/dephosphorylation cycle pathway has been questioned, however, because the well-studied Saccharomyces cerevisiae NMD pathway has not been shown to be regulated by phosphorylation. Here, we used in vitro and in vivo biochemical techniques to show that both S. cerevisiae Upf1p and Upf2p are phosphoproteins. We provide evidence that the phosphorylation of the N-terminal region of Upf2p is crucial for its interaction with Hrp1p, an RNA-binding protein that we previously showed is essential for NMD. We identify specific amino acids in Upf2p's N-terminal domain, including phosphorylated serines, which dictate both its interaction with Hrp1p and its ability to elicit NMD. Our results indicate that phosphorylation of UPF1 and UPF2 is a conserved event in eukaryotes and for the first time provide evidence that Upf2p phosphorylation is crucial for NMD.

A role for CDC7 in repression of transcription at the silent mating-type locus HMR in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (2) ◽  
pp. 1080-1091
Author(s):  
A Axelrod ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Mating Type ◽  
Double Mutant ◽  
Temperature Sensitive ◽  
Alpha Cells ◽  
Nucleotide Substitutions ◽  
Type Locus

The mating-type genes at MAT in Saccharomyces cerevisiae are expressed, whereas the same genes located at HML and HMR are transcriptionally repressed. The DNA element responsible for repression at HMR has been termed a silencer and contains an autonomous replication sequence, a binding site for GRFI/RAPI, and a binding site for ABFI. A double-mutant HMR-E silencer that contains single nucleotide substitutions in both the GRFI/RAPI- and ABFI-binding sites no longer binds either factor in vitro, nor represses transcription at HMR in vivo. In MAT alpha cells, this derepression of a information results in a nonmating phenotype. Second-site suppressor mutations were isolated that restored the alpha mating phenotype to MAT alpha cells containing the double-mutant silencer. One of these suppressors, designated sas1-1, conferred a temperature-sensitive lethal phenotype to the cell. SAS1 was found to be identical to CDC7, a gene which encodes a protein kinase required for the initiation of DNA replication. This new allele of CDC7 was designated cdc7-90. cdc7-90 restored the alpha mating phenotype by restoring silencing. The original allele of CDC7, isolated on the basis of the cell cycle phenotype it confers, also restored silencing, and overexpression of CDC7 interfered with silencing. cdc7-90 did not restore detectable binding of GRFI/RAPI or ABFI to the double-mutant silencer in vitro. These results indicate that a reduced level of CDC7 function restores silencing to a locus defective in binding two factors normally required for silencing.

Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

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