Cognate Polymerase Research Articles

Dynamics of An Archael DNA Polymerase Revealed By Single Molecule Fret

In the archaeon Methanosarcina acetivorans, DNA replication is done by DNA polymerase BI. The processivity of this enzyme is greatly enhanced by a conserved cofactor known as PCNA, which plays a crucial role in orchestrating many replication-related processes. To understand the dynamics of these proteins, we have used single molecule FRET to examine the behavior of PolBI labeled with FRET donor on various DNA structures labeled with FRET acceptor and the effect of PCNA on the dynamics of PolBI. The binding of PolBI to DNA was observed in the low nanomolar concentration range as expected. Interestingly, this polymerase is highly mobile on the DNA structures with two nonadjacent primer strands that are complementary to two different regions in the template strand, 20 nucleotides apart, as evidenced by the frequent transitions between two long-lived FRET states exhibited in single molecule trajectories. To explore the nature of this spontaneous motion, we considered several possible mechanisms including translocations along single- or double-stranded DNA, polymerase binding orientation flipping, and polymerase active site switching. We observed that changes made downstream of the primer/template (P/T) junction have a significant impact on the dynamics of PolBI, indicating the translocation along the single-stranded DNA as the mechanism for the spontaneous motion revealed by FRET. In addition, we have found PCNA not only improves the binding affinity of its cognate polymerase, but suppresses the frequent movement of PolBI from the P/T junction. In summary, many of the dynamics discussed here are reported for the first time and will provide a new perspective for understanding the orchestration of replication-related processes in archaea.

Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures

RNA folding in the cell occurs during transcription. Expedient RNA folding must avoid the formation of undesirable structures as the nascent RNA emerges from the RNA polymerase. We show that efficient folding during transcription of three conserved noncoding RNAs from Escherichia coli, RNase P RNA, signal-recognition particle RNA, and tmRNA is facilitated by their cognate polymerase pausing at specific locations. These pause sites are located between the upstream and downstream portions of all of the native long-range helices in these noncoding RNAs. In the paused complexes, the nascent RNAs form labile structures that sequester these upstream portions in a manner to possibly guide folding. Both the pause sites and the secondary structure of the nonnative portions of the paused complexes are phylogenetically conserved among gamma-proteobacteria. We propose that specific pausing-induced structural formation is a general strategy to facilitate the folding of long-range helices. This polymerase-based mechanism may result in portions of noncoding RNA sequences being evolutionarily conserved for efficient folding during transcription.

Nucleotides at the extremities of the viral RNA of influenza C virus are involved in type-specific interactions with the polymerase complex.

Influenza A and C viruses share common sequences in the terminal noncoding regions of the viral RNA segments. Differences at the 5'- and 3'-ends exist, however, that could contribute to the specificity with which the transcription/replication signals are recognized by the cognate polymerase complexes. Previously, by making use of a transient expression system for the transcription and replication of a reporter RNA template bearing either type A or type C extremities, it was shown that a type C RNA template is transcribed and replicated with equal efficiency by either the type A or the type C polymerase complex, whereas a type A RNA template is less efficiently transcribed and replicated by the type C polymerase complex than by the type A complex. To explore the contribution of the nucleotides at the extremities of the RNAs to this type-specificity, the effect of mutations introduced either alone or in combination at nucleotide 5 at the 3'-end and at nucleotides 3', 6' or 8' at the 5'-end of type A or C RNA templates were studied in the presence of either the type A or the type C polymerase complex. The results indicate that the nature of nucleotides 5 and 6' contribute to type-specificity. Moreover, these results underline the importance of the base pairing between nucleotide 3' and 8' at the 5'-end of the RNA. Thus, it could be suggested that the nature of the nucleotides as well as the stability of the secondary structure at the extremities of the viral RNA are important determinants of type-specificity.

Persistent Yeast Single-stranded RNA Viruses Exist in Vivo as Genomic RNA·RNA Polymerase Complexes in 1:1 Stoichiometry

Yeast narnavirus 20 S and 23 S RNAs encode RNA-dependent RNA polymerases p91 and p104, respectively, but do not encode coat proteins. Both RNAs form ribonucleoprotein complexes with their cognate polymerases. Here we show that these complexes are not localized in mitochondria, unlike the closely related mitoviruses, which reside in these organelles. Cytoplasmic localization of these polymerases was demonstrated by immunofluorescence and by fluorescence emitted from green fluorescent protein-fused polymerases. These fusion proteins were able to form ribonucleoprotein complexes as did the wild-type polymerases. Fluorescent observations and cell fractionation experiments suggested that the polymerases were stabilized by complex formation with their viral RNA genomes. Immunoprecipitation experiments with anti-green fluorescent protein antibodies demonstrated that a single polymerase molecule binds to a single viral RNA genome in the complex. Moreover, the majority (if not all) of 20 S and 23 S RNA molecules were found to form complexes with their cognate RNA polymerases. Since these viral RNAs were not encapsidated, ribonucleoprotein complex formation with their cognate RNA polymerases appears to be their strategy to survive in the host as persistent viruses.

Purification and Characterization of an RNA Polymerase II Phosphatase from Yeast

RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit. An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by >30,000-fold. The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa. The properties of the yeast CTD phosphatase are similar to those of a previously characterized CTD phosphatase from HeLa cells. These properties include stimulation by the general transcription factor IIF (TFIIF), competitive inhibition by RNAP II, magnesium dependence, and resistance to okadaic acid. Both the HeLa and yeast CTD phosphatases are highly specific for their cognate polymerases. Neither phosphatase functions upon the polymerase molecule from the other species, even though the heptapeptide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are essentially identical. The activity of the highly purified CTD phosphatase is stimulated >300-fold by a partially purified fraction of TFIIF. Recombinant TFIIF did not substitute for the TFIIF fraction, indicating that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity. These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is likely composed of at least two components, one of which is 100 and/or 103 kDa.

Prokaryotic transcriptional enhancers and enhancer-binding proteins

A number of prokaryotic enhancer-binding proteins activate transcription by specialized forms of RNA polymerase. The enhancer-binding proteins catalyse isomerization of the initial complex formed between RNA polymerase and a promoter from the closed to the open state. To do so, one class of enhancer-binding proteins contacts its cognate polymerase by DNA loop formation but the other, which is represented by a single member, does not. Despite this difference, both classes of enhancer-binding proteinsmust hydrolyse ATP to catalyse open complex formation.