Biomedical subjects

Anton Meinhart

Publications and source records attributed to Anton Meinhart.

8 recordsLinked to original sources

PubMed2005

A structural perspective of CTD function.

The C-terminal domain (CTD) of RNA polymerase II (Pol II) integrates nuclear events by binding proteins involved in mRNA biogenesis. CTD-binding proteins recognize a specific CTD phosphorylation pattern, which changes during the transcription cycle, due to the action of CTD-modifying enzymes. Structural and functional studies of CTD-binding and -modifying proteins now reveal some of the mechanisms underlying CTD function. Proteins recognize CTD phosphorylation patterns either directly, by contacting phosphorylated residues, or indirectly, without contact to the phosphate. The catalytic mechanisms of CTD kinases and phosphatases are known, but the basis for CTD specificity of these enzymes remains to be understood.

Amino Acid Sequence↗

PubMed2004

Structures of complete RNA polymerase II and its subcomplex, Rpb4/7.

We determined the x-ray structure of the RNA polymerase (Pol) II subcomplex Rpb4/7 at 2.3 A resolution, combined it with a previous structure of the 10-subunit polymerase core, and refined an atomic model of the complete 12-subunit Pol II at 3.8-A resolution. Comparison of the complete Pol II structure with structures of the Pol II core and free Rpb4/7 shows that the core-Rpb4/7 interaction goes along with formation of an alpha-helix in the linker region of the largest Pol II subunit and with folding of the conserved Rpb7 tip loop. Details of the core-Rpb4/7 interface explain facilitated Rpb4/7 dissociation in a temperature-sensitive Pol II mutant and specific assembly of Pol I with its Rpb4/7 counterpart, A43/14. The refined atomic model of Pol II serves as the new reference structure for analysis of the transcription mechanism and enables structure solution of complexes of the complete enzyme with additional factors and nucleic acids by molecular replacement.

Amino Acid Sequence↗

PubMed2004

Structure and mechanism of RNA polymerase II CTD phosphatases.

Recycling of RNA polymerase II (Pol II) after transcription requires dephosphorylation of the polymerase C-terminal domain (CTD) by the phosphatase Fcp1. We report the X-ray structure of the small CTD phosphatase Scp1, which is homologous to the Fcp1 catalytic domain. The structure shows a core fold and an active center similar to those of phosphotransferases and phosphohydrolases that solely share a DXDX(V/T) signature motif with Fcp1/Scp1. We demonstrate that the first aspartate in the signature motif undergoes metal-assisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate that was structurally mimicked with the inhibitor beryllofluoride. Specificity may result from CTD binding to a conserved hydrophobic pocket between the active site and an insertion domain that is unique to Fcp1/Scp1. Fcp1 specificity may additionally arise from phosphatase recruitment near the CTD via the Pol II subcomplex Rpb4/7, which is shown to be required for binding of Fcp1 to the polymerase in vitro.

Amino Acid Sequence↗

PubMed2004

Recognition of RNA polymerase II carboxy-terminal domain by 3'-RNA-processing factors.

During transcription, RNA polymerase (Pol) II synthesizes eukaryotic messenger RNA. Transcription is coupled to RNA processing by the carboxy-terminal domain (CTD) of Pol II, which consists of up to 52 repeats of the sequence Tyr 1-Ser 2-Pro 3-Thr 4-Ser 5-Pro 6-Ser 7 (refs 1, 2). After phosphorylation, the CTD binds tightly to a conserved CTD-interacting domain (CID) present in the proteins Pcf11 and Nrd1, which are essential and evolutionarily conserved factors for polyadenylation-dependent and -independent 3'-RNA processing, respectively. Here we describe the structure of a Ser 2-phosphorylated CTD peptide bound to the CID domain of Pcf11. The CTD motif Ser 2-Pro 3-Thr 4-Ser 5 forms a beta-turn that binds to a conserved groove in the CID domain. The Ser 2 phosphate group does not make direct contact with the CID domain, but may be recognized indirectly because it stabilizes the beta-turn with an additional hydrogen bond. Iteration of the peptide structure results in a compact beta-spiral model of the CTD. The model suggests that, during the mRNA transcription-processing cycle, compact spiral regions in the CTD are unravelled and regenerated in a phosphorylation-dependent manner.

Amino Acid Motifs↗

PubMed2003

An extended winged helix domain in general transcription factor E/IIE alpha.

Initiation of eukaryotic mRNA transcription requires melting of promoter DNA with the help of the general transcription factors TFIIE and TFIIH. Here we define a conserved and functionally essential N-terminal domain in TFE, the archaeal homolog of the large TFIIE subunit alpha. X-ray crystallography shows that this TFE domain adopts a winged helix-turn-helix (winged helix) fold, extended by specific alpha-helices at the N and C termini. Although the winged helix fold is often found in DNA-binding proteins, we show that TFE is not a typical DNA-binding winged helix protein, because its putative DNA-binding face shows a negatively charged groove and an unusually long wing, and because the domain lacks DNA-binding activity in vitro. The groove and a conserved hydrophobic surface patch on the additional N-terminal alpha-helix may, however, allow for interactions with other general transcription factors and RNA polymerase. Homology modeling shows that the TFE domain is conserved in TFIIE alpha, including the potential functional surfaces.

Amino Acid Sequence↗

PubMed2003

The mRNA transcription/processing factor Ssu72 is a potential tyrosine phosphatase.

Ssu72 is an essential and highly conserved protein involved in mRNA transcription and 3'-end processing. The biochemical function of Ssu72 was so far unknown. We report here evidence that Ssu72 is a phosphatase that resembles protein tyrosine phosphatases (PTPases). First, recombinant Ssu72 cleaves the phosphotyrosine analogue p-nitrophenylphosphate, and this catalytic activity is impaired by PTPase-inhibiting agents. Second, the Ssu72 sequence contains the CX(5)R signature motif of PTPases; mutation of the catalytic cysteine in this motif abolishes Ssu72 activity in vitro and has been shown to confer lethality in vivo. Third, secondary structure prediction and site-directed mutagenesis predict that Ssu72 adopts the fold of PTPases of the low molecular weight family. Distinguishing features, such as a short "aspartate loop" at the active site, suggest however that Ssu72 is the founding member of a new phosphatase subfamily. The novel Ssu72 activity may regulate coupling events during mRNA biogenesis.

Amino Acid Sequence↗

PubMed2003

Crystal structure of the plasmid maintenance system epsilon/zeta: functional mechanism of toxin zeta and inactivation by epsilon 2 zeta 2 complex formation.

Programmed cell death in prokaryotes is frequently found as postsegregational killing. It relies on antitoxin/toxin systems that secure stable inheritance of low and medium copy number plasmids during cell division and kill cells that have lost the plasmid. The broad-host-range, low-copy-number plasmid pSM19035 from Streptococcus pyogenes carries the genes encoding the antitoxin/toxin system epsilon/zeta and antibiotic resistance proteins, among others. The crystal structure of the biologically nontoxic epsilon(2)zeta(2) protein complex at a 1.95-A resolution and site-directed mutagenesis showed that free zeta acts as phosphotransferase by using ATPGTP. In epsilon(2)zeta(2), the toxin zeta is inactivated because the N-terminal helix of the antitoxin epsilon blocks the ATPGTP-binding site. To our knowledge, this is the first prokaryotic postsegregational killing system that has been entirely structurally characterized.

Amino Acid Sequence↗

PubMed2002

In vitro and in vivo stability of the epsilon2zeta2 protein complex of the broad host-range Streptococcus pyogenes pSM19035 addiction system.

Streptococcus pyogenes pSM19035-encoded epsilon (10.7 kDa) and zeta (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with zeta acting as toxin that kills plasmid-deprived cells and epsilon as an antitoxin that neutralises the activity of zeta. The epsilon and zeta proteins co-purify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as epsilon2zeta2 heterotetramer in solution. Co-crystals of the epsilon2zeta2 complex contain epsilon and zeta in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the zeta protein has a significantly lower thermodynamic stability than the epsilon protein both in free state and in the complex. Proteolytic studies indicate that zeta protein is more stable in the epsilon2zeta2 complex than in the free state. In vivo studies reveal a short half-life of the epsilon antitoxin (-18 min) and a long lifetime of the zeta toxin (>60 min). When transcription-translation of a plasmid containing the epsilon and zeta genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the epsilon protein from the background.

Algorithms↗