摘要:

mRNA capping entails GMP transfer from GTP to a 5′ diphosphate RNA end to form the structure G(5′)ppp(5′)N. A similar reaction involving AMP transfer to the 5′ monophosphate end of DNA or RNA occurs during strand joining by polynucleotide ligases. In both cases, nucleotidyl transfer occurs through a covalent lysyl‐NMP intermediate. Sequence conservation among capping enzymes and ATP‐dependent ligases in the vicinity of the active site lysine (KxDG) and at five other co‐linear motifs suggests a common structural basis for covalent catalysis. Mutational studies support this view. We propose that the cellular and DNA virus capping enzymes and ATP‐dependent ligases constitute a protein superfamily evolved from a common ancestral enzyme. Within this superfamily, the cellular capping enzymes display more extensive similarity to the ligases than they do to the poxvirus capping enzymes. Recent studies suggest that eukaryotic RNA viruses have evolved alternative pathways of cap metabolism catalysed by structurally unrelated enzymes that nonetheless employ a phosphoramidate intermediate. Comparative analysis of these enzymes, particularly at the structural level, should illuminate the shared reaction mechanism while clarifying the basis for nucleotide specificity and end recognition. The capping enzymes merit close attention as potential targets for an

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030405.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

RNase P is responsible for the maturation of the 5′‐termini of tRNA molecules in all cells studied to date. This ribonucleoprotein has to recognize and identify its cleavage site on a large number of different precursors. This review covers what is currently known about the function of the catalytic subunit ofEscherichia coliRNase P, M1 RNA, and the protein subunit, C5, in particular with respect to cleavage‐site selection. Recent genetic and biochemical data show that the two C residues in the 3′‐terminal CCA sequence of a precursor interact with the enzyme through Watson‐Crick base‐pairing. This is suggested to result in unfolding of the amino acid acceptor‐stem and exposure of the cleavage site. Furthermore, other close contact points between M1 RNA and its substrate have recently been identified. These data, together with the two existing three‐dimensional structure models of M1 RNA in complex with its substrate, establish a platform that will enable us to seek an understanding of the underlying mechanism of cleavage by th

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030411.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

This review discusses the phenomenon of recycling of cell wall peptides. It is a major, though non‐essential, pathway of the cell and is required for induction of β‐lactamase. Consequently, the recycling pathway is viewed as a possible signalling vehicle, informing the cell of the condition of the murein sacculus, an essential structure existing outside the cell itself. As the study of this phenomenon is in its infancy, several speculations are offered for a possible regulatory func

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030421.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Lantibiotics form a family of highly modified peptides which are secreted by several Gram‐positive bacteria. They exhibit antimicrobial activity, mainly against other Gram‐positive bacteria, by forming pores in the cellular membrane. These antimicrobial peptides are ribosomally synthesized and contain leader peptides which do not show the characteristics of signal sequences. Several amino acid residues of the precursor lantibiotic are enzymatically modified, whereafter secretion and processing of the leader peptide takes place, yielding the active antimicrobial substance. For several lantibiotics the gene clusters encoding biosynthetic enzymes, translocator proteins, self‐protection proteins, processing enzymes and regulatory proteins have been identified. ThisMicroReviewdescribes the current knowledge about the biosynthetic, immunity and regulatory processes leading to lantibiotic production. Most of the attention is focused on the lantibiotic nisin, which is produced by the food‐grade bacteriumLactococcus lactisand is widely used as a preservative in the food i

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030427.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Mutations in therec15gene ofSchizosaccharomyces pombereduce meiotic recombinant frequencies, in the three intervals tested, as much as 1000‐fold but have no detectable mitotic phenotype. Therec15gene was mapped to within 1 cM ofmat1. The gene was cloned by genetic complementation and its nucleotide sequence determined. Deletion analysis and gene replacement confirmed that the clones contained therec15gene on DNA fragments as short as 1.3 kb. The nucleotide sequence of the 1.3 kb fragment predicted thatrec15had a 49 bp intron separating two exons encoding a 180‐amino‐acid polypeptide product. This predicted intron was confirmed by reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis. During thermally induced meiosis in apat1–114(Ts) mutant, therec15transcripts were induced to maximal levels at 2–3 h but were present at much lower levels before and after this time. The transient induction of the transcripts and the phenotype of arec15null (deletion) mutation suggest that therec15gene product is required during the early stages of meiosis for meiotic

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030439.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

A set of gene fusions was constructed between the pehA gene encoding the secreted endopolygalacturonase (PehA) and theblagene coding for a normally periplasmic β‐lactamase (Bla). The resulting hybrid proteins were specifically and actively routed out of the cells via the Out‐terminal branch of the general secretory pathway (GSP) inErwinia carotovorasubsp.carotovora (Ecc), provided that no more than the last two amino acids (aa) of the PehA domain were excluded from the fusion. However, both PehA‐Bla hybrid proteins and PehA variants lacking at least four aa from the C‐terminus of the PehA accumulated in the periplasm. Also, overexpression of the gene fusions prevented extracellular targeting of the hybrid proteins. Site‐directed mutagenesis of the codons −4 and −3 (encoding Asn‐373 and Val‐374, respectively) from the end of the pehA gene and analysis of the protein products suggested that the Val‐374 was important both for the structure and secretion of PehA, while the Asn‐373 proved to be insignificant. We conclude that: (i) the GSP of Ecc is capable of secreting heterologous proteins; (ii) as the PehA protein can accommodate C‐terminal extensions, secretion can occur with no part of the proposed targeting signal lying within the C‐terminal extremity of a secreted molecule; and (iii) residues within the C‐terminus of PehA play a role in secretion, possibly through stabilization of a structure needed for proper exposition of

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030449.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Entry ofShigella flexneriinto epithelial cells involves secretory proteins, the lpa proteins, and their dedicated secretion apparatus, the Mxi—Spa translocon, which is encoded by themxiandspaoperons. We have characterized themxiGgene that is located at the proximal part of themxioperon. Inactivation ofmxiGabolished lpa secretion, which indicates that MxiG is an essential component of the Mxi‐Spa translocon. Immunoblotting analysis of membrane fractions suggests that the 42 kDa MxiG protein is associated with both the inner and outer membranes. Taking advantage of the complementation of themxiGmutant by a plasmid carrying a wild‐type copy ofmxiG(which restored lpa secretion, entry into HeLa cells, and cell‐to‐cell spread) we mutagenized themxiGgene carried by the complementing plasmid to replace the RGD motif of MxiG by RAD. This mutation (mxiG*), which had no effect on the stability of the protein, did not affect lpa secretionin vitroor entry into HeLa cells, but impaired intercellular dissemination. Therefore, MxiG and possibly proteins secreted by the Mxi‐Spa translocon are involved not only in entry but also in spread ofshigellabetween epith

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030461.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

InEscherichia coli, TyrR represses and activates transcription of operons required for tyrosine, phenylalanine and tryptophan biosynthesis and uptake. The TyrR central domain is homologous with NtrC and some other bacterial regulatory proteins, although TyrR regulates σ70, not σ54, promoters. We isolated a central domain TyrR mutant (TyrR E274Q) by substitution of a normally conserved amino acid. The mutant was unable to bring about tyrosine‐mediated repression ofaroF, aroL, tyrB, andtyrPand had diminished capability for tyrosine‐ and phenylalanine‐mediated repression ofaroP. In contrast, it was able to effect wild‐type levels of phenylalanine‐mediated repression ofaroG, tryptophan‐mediated repression ofaroPand transcriptional activation ofmtrandtyrP. The binding of purified TyrR E274Q to ATP (a requirement for tyrosine binding) and to the strong TyrR box oftyrPoperator DNA were normal, but tyrosine binding and tyrosine‐dependent hexamerization were significantly impaired. These properties are consistent with the proposal that self association is essential for tyrosine‐mediated repression by TyrR but not for tyrosine‐ or phenylalanine‐mediated activation. E274of TyrR must participate in either the binding of tyrosine, or the coupling of ATP binding with a conformational change that alters the affinity of the ATP‐dependent aromatic

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030471.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

InEscherichia coli, genetic regulation of aromatic amino acid biosynthesis and uptake is effected by the protein TyrR, which acts via ligand‐mediated repression and activation. Characterization of the interactions of tyrosine, phenylalanine and tryptophan with TyrR revealed the presence of two separate aromatic amino acid‐binding sites, one ATP‐dependent, the other ATP‐independent. Binding to the ATP‐dependent site induces the self‐association of TyrR. Using sedimentation equilibrium analyses, dissociation constants for this site in the dimeric and hexameric forms of TyrR were determined to be 330 μM and 24 μM, respectively, for tyrosine, and 55 mM and 3.7 mM, respectively, for phenylalanine. Tryptophan bound with a strength similar to that of phenylalanine, and both phenylalanine and tryptophan competed with the binding of tyrosine. The ATP‐independent site, which has not been observed previously, was characterized by ultraviolet (u.v.) difference spectroscopy and a sedimentation‐velocity meniscus‐depletion method. Phenylalanine bound co‐operatively to this site, exhibiting half‐saturation at 260 µM. Tryptophan competed weakly with phenylalanine, half‐saturation occurring at 1.2 mM. No binding of tyrosine to this site could be detected. We propose that the binding of phenylalanine or tryptophan to this ATP‐independent site is responsible for phenylalanine‐ and tryptoph

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030483.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Characterization of functions that render DNA susceptible to rearrangement is important for a better understanding of genome instability. In a previous work, we showed that sequences located downstream of a strong promoter are particularly prone to deletion. In this paper, the parameters that influence transcription‐induced deletions were studied. pBR322 derivatives carrying the M13 (+) replication origin and a PTac‐dependent transcription region were used. Deletion formation was analysed in the presence of the replication protein of M13, which introduces a nick at the phage replication origin, and in arep−strain to avoid M13‐driven replication. Our study showed that: (i) 4 h after induction of transcription, a few per cent of the plasmids have experienced a deletion; (ii) these deletions result in joining of the M13 replication origin to a nucleotide located in or downstream of the transcribed region; (iii) deletion formation strongly depends on the orientation of transcription, on promoter strength and transcript length, but is independent of translation; (iv) formation of transcription‐induced supercoiling domains does not induce deletion formation. We propose that deletions in the transcribed region result from collisions between converging replication and transcription ma

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17030493.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY