Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state.
Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (I(C)). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of I(C) through a more extended state associated with the membrane surface (I(S)). While I(C) appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c. (+info
The magnitude of changes in guanidine-HCl unfolding m-values in the protein, iso-1-cytochrome c, depends upon the substructure containing the mutation.
Hydrophilic to hydrophobic mutations have been made at 11 solvent exposed sites on the surface of iso-1-cytochrome c. Most of these mutations involve the replacement of lysine with methionine, which is nearly isosteric with lysine. Minimal perturbation to the native structure is expected, and this expectation is confirmed by infrared amide I spectroscopy. Guanidine hydrochloride denaturation studies demonstrate that these variants affect the magnitude of the m-value, the rate of change of free energy with respect to denaturant concentration, to different degrees. Changes in m-values are indicative of changes in the equilibrium folding mechanism of a protein. Decreases in m-values are normally thought to result either from an increased population of intermediates during unfolding or from a more compact denatured state. When cytochrome c is considered in terms of its thermodynamic substructures, the changes in the m-value for a given variant appear to depend upon the substructure in which the mutation is made. These data indicate that the relative stabilities and physical properties of substructures of cytochrome c play an important determining role in the equilibrium folding mechanism of this protein. (+info
A mutation in GRS1, a glycyl-tRNA synthetase, affects 3'-end formation in Saccharomyces cerevisiae.
3'-end formation is a complex and incompletely understood process involving both cis-acting and trans-acting factors. As part of an effort to examine the mechanisms of transcription termination by RNA polymerase II, a mutant hunt for strains defective in 3'-end formation was conducted. Following random mutagenesis, a temperature-sensitive strain exhibiting several phenotypes consistent with a role in transcription termination was isolated. First, readthrough of a terminator increases significantly in the mutant strain. Accordingly, RNA analysis indicates a decrease in the level of terminated transcripts, both in vivo and in vitro. Moreover, a plasmid stability assay in which high levels of readthrough lead to high levels of plasmid loss and transcription run-on analysis also demonstrate defective termination of transcription. Examination of polyadenylation and cleavage by the mutant strain indicates these processes are not affected. These results represent the first example of a transcription termination factor in Saccharomyces cerevisiae that affects transcription termination independent of 3'-end processing of mRNA. Complementation studies identified GRS1, an aminoacyl-tRNA synthetase, as the complementing gene. Sequence analysis of grs1-1 in the mutant strain revealed that nucleotides 1656 and 1657 were both C to T transitions, resulting in a single amino acid change of proline to phenylalanine. Further studies revealed GRS1 is essential, and the grs1-1 allele confers the temperature-sensitive growth defect associated with the mutant strain. Finally, we observed structures with some similarity to tRNA molecules within the 3'-end of various yeast genes. On the basis of our results, we suggest Grs1p is a transcription termination factor that may interact with the 3'-end of pre-mRNA to promote 3'-end formation. (+info
Oxygen sensing in yeast: evidence for the involvement of the respiratory chain in regulating the transcription of a subset of hypoxic genes.
Oxygen availability affects the transcription of a number of genes in nearly all organisms. Although the molecular mechanisms for sensing oxygen are not precisely known, heme is thought to play a pivotal role. Here, we address the possibility that oxygen sensing in yeast, as in mammals, involves a redox-sensitive hemoprotein. We have found that carbon monoxide (CO) completely blocks the anoxia-induced expression of two hypoxic genes, OLE1 and CYC7, partially blocks the induction of a third gene, COX5b, and has no effect on the expression of other hypoxic or aerobic genes. In addition, transition metals (Co and Ni) induce the expression of OLE1 and CYC7 in a concentration-dependent manner under aerobic conditions. These findings suggest that the redox state of an oxygen-binding hemoprotein is involved in controlling the expression of at least two hypoxic yeast genes. By using mutants deficient in each of the two major yeast CO-binding hemoproteins (cytochrome c oxidase and flavohemoglobin), respiratory inhibitors, and cob1 and rho0 mutants, we have found that the respiratory chain is involved in the anoxic induction of these two genes and that cytochrome c oxidase is likely the hemoprotein "sensor." Our findings also indicate that there are at least two classes of hypoxic genes in yeast (CO sensitive and CO insensitive) and imply that multiple pathways/mechanisms are involved in modulating the expression of hypoxic yeast genes. (+info
Pseudo-native motifs in the noncovalent heme-apocytochrome c complex. Evidence from antibody binding studies by enzyme-linked immunosorbent assay and microcalorimetry.
When beef heart apocytochrome c is unfolded, it folds upon noncovalent heme binding (Dumont, M. E., Corin, A. F., and Campbell, G.A. (1994) Biochemistry, 33, 7368-7378). Here, the conformation of the heme-apocytochrome noncovalent complex is compared with that of holocytochrome c. A purification method was designed for obtaining in large amounts apocytochrome c that was shown by amino acid analysis and mass spectroscopy to be chemically intact. The apoprotein and its noncovalent complex were characterized by absorption, fluorescence, circular dichroism, and sedimentation velocity, confirming previous reports. Sedimentation-diffusion equilibrium showed that the apoprotein and its noncovalent complex with heme were monomeric. Surprisingly, whereas apocytochrome c was quite soluble, the noncovalent complex slowly formed heavy aggregates, thus precluding experiments at the concentrations needed for structural studies. Two monoclonal antibodies that bind strongly to distinct antigenic sites on native holocytochrome were used to probe the noncovalent complex conformation. For both antibodies, the affinity for the noncovalent complex was only about 5-10-fold smaller than that for native holocytochrome c, and about 50-100-fold larger than that for apocytochrome c. These results indicate that the noncovalent complex, although not entirely native, carries some pseudo-native structural motifs. (+info
Rational design of a more stable yeast iso-1-cytochrome c.
Yeast iso-1-cytochrome c is one of the least stable mitochondrial cytochromes c. We have used a coordinated approach, combining the known functional and structural properties of cytochromes c, to engineer mutations into yeast iso-1-cytochrome c with the goal of selectively increasing the stability of the protein. The two redox forms of the native protein and six different mutant forms of yeast iso-1-cytochrome c were analyzed by differential scanning calorimetry (DSC). The relative stability, expressed as the difference in the Gibb's free energy of denaturation at a given temperature between the native and mutant forms (DeltaDeltaG(Tref)), was determined for each of the proteins. In both oxidation states, the mutant proteins C102T, T69E/C102T, T96A/C102T, and T69E/T96A/C102T were more stable than the wild-type protein, respectively. The increased stability of the mutant proteins is proposed to be due to the removal of a rare surface cysteine and the stabilization of two distorted alpha-helices. (+info
Post-transcriptional adenylation of signal recognition particle RNA is carried out by an enzyme different from mRNA Poly(A) polymerase.
A fraction of the signal recognition particle (SRP) RNA from human, rat, Xenopus, and Saccharomyces cerevisiae cells contains a single post-transcriptionally added adenylic acid residue on its 3'-end; in the case of human SRP RNA, over 60% of the SRP RNA molecules contain a nontemplated adenylic acid residue on their 3'-ends (Sinha, K. M., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). In this study, we investigated the enzyme that is involved in this 3'-end adenylation of SRP RNA. A U1A protein peptide conjugated to albumin completely inhibited the polyadenylation of a SV40 mRNA by HeLa cell nuclear extract in vitro; however, the 3'-end adenylation of human SRP RNA or Alu RNA, which corresponds to 5' and 3'-ends of SRP RNA, was not affected by this U1A peptide conjugate. SRP RNA from mutant strains of S. cerevisiae with a temperature-sensitive mRNA poly(A) polymerase grown at a restrictive temperature of 37 degrees C also contained a post-transcriptionally added adenylic acid residue just like SRP RNA from wild-type cells and mutant cells grown at permissive temperature of 23 degrees C. In addition, binding of SRP 9/14-kDa protein heterodimer was required for adenylation of Alu RNA in vitro. These lines of evidence, along with other data, show that post-transcriptional adenylation of SRP and Alu RNAs is carried out by a novel enzyme that is distinct from the mRNA poly(A) polymerase, CCA-adding enzyme, and nonspecific terminal transferase. (+info
Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisiae.
N-terminal acetylation can occur cotranslationally on the initiator methionine residue or on the penultimate residue if the methionine is cleaved. We investigated the three N-terminal acetyltransferases (NATs), Ard1p/Nat1p, Nat3p and Mak3p. Ard1p and Mak3p are significantly related to each other by amino acid sequence, as is Nat3p, which was uncovered in this study using programming alignment procedures. Mutants deleted in any one of these NAT genes were viable, but some exhibited diminished mating efficiency and reduced growth at 37 degrees C, and on glycerol and NaCl-containing media. The three NATs had the following substrate specificities as determined in vivo by examining acetylation of 14 altered forms of iso-1-cytochrome c and 55 abundant normal proteins in each of the deleted strains: Ard1p/Nat1p, subclasses with Ser-, Ala-, Gly- and Thr-termini; Nat3p, Met-Glu- and Met-Asp- and a subclass of Met-Asn-termini; and Mak3p subclasses with Met-Ile- and Met-Leu-termini. In addition, a special subclass of substrates with Ser-Glu- Phe-, Ala-Glu-Phe- and Gly-Glu-Phe-termini required all three NATs for acetylation. (+info