Alfredo Hernandez

Regulatory Interactions between RNA and Polycomb Repressive Complex 2

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that is localized to thousands of mammalian genes. Though important to human disease and as a drug target, how PRC2 is recruited remains unclear. One model invokes cis-regulatory RNA. Herein, we biochemically and functionally probe PRC2s recognition of RNA using the X-inactivation model. We observe surprisingly high discriminatory capabilities. While SUZ12 and JARID2 subunits can bind RNA, EZH2 has highest affinity and is somewhat promiscuous. EED regulates the affinity of EZH2 for RNA, lending greater specificity to PRC2-RNA interactions. Intriguingly, while RNA is crucial for targeting, RNA inhibits EZH2s catalytic activity. JARID2 weakens PRC2s binding to RNA and relieves catalytic inhibition. We propose that RNA guides PRC2 to its target but inhibits its enzymatic activity until PRC2 associates with JARID2 on chromatin. Our study provides a molecular view of regulatory interactions between RNA and PRC2 at the chromatin interface.

REH2 RNA helicase in kinetoplastid mitochondria: ribonucleoprotein complexes and essential motifs for unwinding and guide RNA (gRNA) binding.

Regulation of gene expression in kinetoplastid mitochondria is largely post-transcriptional and involves the orchestration of polycistronic RNA processing, 3′-terminal maturation, RNA editing, turnover, and translation; however, these processes remain poorly studied. Core editing complexes and their U-insertion/deletion activities are relatively well characterized, and a battery of ancillary factors has recently emerged. This study characterized a novel DExH-box RNA helicase, termed here REH2 (RNA editing associated helicase 2), in unique ribonucleoprotein complexes that exhibit unwinding and guide RNA binding activities, both of which required a double-stranded RNA-binding domain (dsRBD) and a functional helicase motif I of REH2. REH2 complexes and recently identified related particles share a multiprotein core but are distinguished by several differential polypeptides. Finally, REH2 associates transiently, via RNA, with editing complexes, mitochondrial ribosomes, and several ancillary factors that control editing and RNA stability. We propose that these putative higher order structures coordinate mitochondrial gene expression.

The RNA Polymerase of Marine Cyanophage Syn5

Background: Cyanophages are dominant viruses in the ocean while little has been known on their nucleic acid metabolism. Results: The RNA polymerase of cyanophage Syn5 has been purified and characterized and the Syn5 promoters identified. Conclusion: The Syn5 RNA polymerase and promoters have unique and ocean-adapted features. Significance: The first characterized single-subunit RNA polymerase from marine organisms. A single subunit DNA-dependent RNA polymerase was identified and purified to apparent homogeneity from cyanophage Syn5 that infects the marine cyanobacteria Synechococcus. Syn5 is homologous to bacteriophage T7 that infects Escherichia coli. Using the purified enzyme its promoter has been identified by examining transcription of segments of Syn5 DNA and sequencing the 5′-termini of the transcripts. Only two Syn5 RNAP promoters, having the sequence 5′-ATTGGGCACCCGTAA-3′, are found within the Syn5 genome. One promoter is located within the Syn5 RNA polymerase gene and the other is located close to the right genetic end of the genome. The purified enzyme and its promoter have enabled a determination of the requirements for transcription. Unlike the salt-sensitive bacteriophage T7 RNA polymerase, this marine RNA polymerase requires 160 mm potassium for maximal activity. The optimal temperature for Syn5 RNA polymerase is 24 °C, much lower than that for T7 RNA polymerase. Magnesium is required as a cofactor although some activity is observed with ferrous ions. Syn5 RNA polymerase is more efficient in utilizing low concentrations of ribonucleotides than T7 RNA polymerase.

Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase

The substitution of 2′-fluoro for 2′-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and applications involving RNA. We report that the RNA polymerase from the marine cyanophage Syn5 has an intrinsic low discrimination against the incorporation of 2′-fluoro dNMPs during transcription elongation. The presence of both magnesium and manganese ions at high concentrations further reduce this discrimination without decreasing the efficiency of incorporation. We have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with phenylalanine (Y564F) that further decreases the discrimination against 2′-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that affect the yield of RNA and incorporation of 2′-fluoro-dNMPs by Syn5 RNA polymerase have been identified.

Substrate Determinants for RNA Editing and Editing Complex Interactions at a Site for Full-round U Insertion

Multisubunit RNA editing complexes catalyze uridylate insertion/deletion RNA editing directed by complementary guide RNAs (gRNAs). Editing in trypanosome mitochondria is transcript-specific and developmentally controlled, but the molecular mechanisms of substrate specificity remain unknown. Here we used a minimal A6 pre-mRNA/gRNA substrate to define functional determinants for full-round insertion and editing complex interactions at the editing site 2 (ES2). Editing begins with pre-mRNA cleavage within an internal loop flanked by upstream and downstream duplexes with gRNA. We found that substrate recognition around the internal loop is sequence-independent and that completely artificial duplexes spanning a single helical turn are functional. Furthermore, after our report of cross-linking interactions at the deletion ES1 (35), we show for the first time editing complex contacts at an insertion ES. Our studies using site-specific ribose 2′ substitutions defined 2′-hydroxyls within the (a) gRNA loop region and (b) flanking helixes that markedly stimulate both pre-mRNA cleavage and editing complex interactions at ES2. Modification of the downstream helix affected scissile bond specificity. Notably, a single 2′-hydroxyl at ES2 is essential for cleavage but dispensable for editing complex cross-linking. This study provides new insights on substrate recognition during full-round editing, including the relevance of secondary structure and the first functional association of specific (pre-mRNA and gRNA) riboses with both endonuclease cleavage and cross-linking activities of editing complexes at an ES. Importantly, most observed cross-linking interactions are both conserved and relatively stable at ES2 and ES1 in hybrid substrates. However, they were also detected as transient low-stability contacts in a non-edited transcript.

Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

Significance Lagging-strand DNA is replicated in multiple segments called Okazaki fragments, whose formation involves a complex molecular cycle mediated by DNA primase, polymerase, and other replisome components. In addition, synthesis of the lagging strand must occur in lockstep with the leading strand. Using the simple replication system of bacteriophage T7, we found that primer release from the DNA primase domain of T7 primase helicase is a critical regulatory event in the initiation of Okazaki fragments and that the T7 single-stranded binding protein, gp2.5, regulates initiation timing. DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers

Significance Most DNA polymerases initiate DNA synthesis by extending a preexisting primer. Exceptions to this dogma are recently characterized bifunctional primase–polymerases (prim–pols) that resemble archaeal primases in their structure and initiate DNA synthesis de novo using only NTPs or dNTPs. We report here a DNA polymerase encoded by a phage NrS-1 from deep-sea vents. NrS-1 has a genome organization unlike any other known phage. Although this polymerase does not contain a zinc-binding motif typical for primases, it is nonetheless able to initiate DNA synthesis from a specific DNA sequence exclusively using dNTPs. Thus, it represents a unique de novo replicative DNA polymerase that possesses features found in DNA polymerases, primases, and RNA polymerases. A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase–polymerases (prim–pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

Modulation of RNA primer formation by Mn(II)-substituted T7 DNA primase

Lagging strand DNA synthesis by DNA polymerase requires RNA primers produced by DNA primase. The N-terminal primase domain of the gene 4 protein of phage T7 comprises a zinc-binding domain that recognizes a specific DNA sequence and an RNA polymerase domain that catalyzes RNA polymerization. Based on its crystal structure, the RNA polymerase domain contains two Mg(II) ions. Mn(II) substitution leads to elevated RNA primer synthesis by T7 DNA primase. NMR analysis revealed that upon binding Mn(II), T7 DNA primase undergoes conformational changes near the metal cofactor binding site that are not observed when the enzyme binds Mg(II). A machine-learning algorithm called linear discriminant analysis (LDA) was trained by using the large collection of Mn(II) and Mg(II) binding sites available in the protein data bank (PDB). Application of the model to DNA primase revealed a preference in the enzyme’s second metal binding site for Mn(II) over Mg(II), suggesting that T7 DNA primase activity modulation when bound to Mn(II) is based on structural changes in the enzyme.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Gp2.5, the multifunctional bacteriophage T7 single-stranded DNA binding protein

The essential bacteriophage T7-encoded single-stranded DNA binding protein is the nexus of T7 DNA metabolism. Multiple layers of macromolecular interactions mediate its function in replication, recombination, repair, and the maturation of viral genomes. In addition to binding ssDNA, the protein binds to DNA polymerase and DNA helicase, regulating their activities. The protein displays potent homologous DNA annealing activity, underscoring its role in recombination.