Transcription start site heterogeneity and its role in RNA fate determination distinguish HIV-1 from other retroviruses and are mediated by core promoter elements.

HIV-1 uses heterogeneous transcription start sites (TSSs) to generate two RNA 5´ isoforms that adopt radically different structures and perform distinct replication functions. Although these RNAs differ in length by only two bases, exclusively, the shorter RNA is encapsidated while the longer RNA is excluded from virions and provides intracellular functions. The current study examined TSS usage and packaging selectivity for a broad range of retroviruses and found that heterogeneous TSS usage was a conserved feature of all tested HIV-1 strains, but all other retroviruses examined displayed unique TSSs. Phylogenetic comparisons and chimeric viruses' properties provided evidence that this mechanism of RNA fate determination was an innovation of the HIV-1 lineage, with determinants mapping to core promoter elements. Fine-tuning differences between HIV-1 and HIV-2, which uses a unique TSS, implicated purine residue positioning plus a specific TSS-adjacent dinucleotide in specifying multiplicity of TSS usage. Based on these findings, HIV-1 expression constructs were generated that differed from the parental strain by only two point mutations yet each expressed only one of HIV-1's two RNAs. Replication defects of the variant with only the presumptive founder TSS were less severe than those for the virus with only the secondary start site. IMPORTANCE Retroviruses use RNA both to encode their proteins and to serve in place of DNA as their genomes. A recent surprising discovery was that the genomic RNAs and messenger RNAs of HIV-1 are not identical but instead differ subtly on one of their ends. These differences enable the functional separation of HIV-1 RNAs into genome and messenger roles. In this report, we examined a broad collection of HIV-1-related viruses and discovered that each produced only one end class of RNA, and thus must differ from HIV-1 in how they specify RNA fates. By comparing regulatory signals, we generated virus variants that pinpointed the determinants of HIV-1 RNA fates, as well as HIV-1 variants that produced only one or the other functional class of RNA. Competition and replication assays confirmed that HIV-1 has evolved to rely on the coordinated actions of both its RNA forms.

Transcription start site heterogeneity and its role in RNA fate determination distinguish HIV-1 from other retroviruses and are mediated by core promoter elements.

HIV-1 uses heterogeneous transcription start sites (TSSs) to generate two RNA 5' isoforms that adopt radically different structures and perform distinct replication functions. Although these RNAs differ in length by only two bases, exclusively the shorter RNA is encapsidated while the longer RNA is excluded from virions and provides intracellular functions. The current study examined TSS usage and packaging selectivity for a broad range of retroviruses and found that heterogenous TSS usage was a conserved feature of all tested HIV-1 strains, but all other retroviruses examined displayed unique TSSs. Phylogenetic csomparisons and chimeric viruses' properties provided evidence that this mechanism of RNA fate determination was an innovation of the HIV-1 lineage, with determinants mapping to core promoter elements. Fine-tuning differences between HIV-1 and HIV-2, which uses a unique TSS, implicated purine residue positioning plus a specific TSS-adjacent dinucleotide in specifying multiplicity of TSS usage. Based on these findings, HIV-1 expression constructs were generated that differed from the parental strain by only two point mutations yet each expressed only one of HIV-1's two RNAs. Replication defects of the variant with only the presumptive founder TSS were less severe than those for the virus with only the secondary start site.

Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction.

Single-stranded telomeric overhangs are ∼200 nucleotides long and can form tandem G-quadruplex (GQ) structures, which reduce their accessibility to nucleases and proteins that activate DNA damage response. Whether these tandem GQs further stack to form compact superstructures, which may provide better protection for longer telomeres, is not known. We report single-molecule measurements where the accessibility of 24-144 nucleotide long human telomeric DNA molecules is interrogated by a short PNA molecule that is complementary to a single GGGTTA repeat, as implemented in the FRET-PAINT method. Binding of the PNA strand to available GGGTTA sequences results in discrete FRET bursts which were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. The binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3'- or 5'-ends, suggesting these regions are more accessible. Significantly, the binding frequency per telomeric repeat monotonically decreased with increasing telomere length. These results are consistent with telomeres forming more compact structures at longer lengths, reducing accessibility of these critical genomic sites.

Ribosomal RNA Methyltransferase RsmC Moonlights as an RNA Chaperone.

Ribosomes are ribonucleoprotein particles that are essential for protein biosynthesis in all forms of life. During ribosome biogenesis, transcription, folding, modification, and processing of rRNA are coupled to the assembly of proteins. Various assembly factors are required to synchronize all different processes that occur during ribosome biogenesis. Herein, the RNA chaperone and RNA strand annealing activity of rRNA modification enzyme ribosome small subunit methyltransferase C (RsmC), which modifies guanine to 2-methylguanosine (m2 G) at position 1207 of 16S rRNA (Escherichia coli nucleotide numbering) located at helix 34 (h34), are reported. A 25-fold increase in the h34 RNA strand annealing rates is observed in the presence of RsmC. Single-molecule FRET experiments confirmed the ability of protein RsmC to denature a non-native structure formed by one of the two h34 strands and to form a native-like duplex. This observed RNA chaperone activity of protein RsmC might play a vital role in the rapid generation of functional ribosomes.

Impact of Small Molecules on Intermolecular G-Quadruplex Formation.

We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC3) on intermolecular G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that had 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures, has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with Pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG repeat case. By demonstrating detection of i-GQ formation at the single molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.

Discovery of a novel small molecular peptide that disrupts helix 34 of bacterial ribosomal RNA.

Despite the advances in modern medicine, antibiotic resistance is a persistent and growing threat to the world. Thus, the discovery and development of novel antibiotics have become crucial to combat multi-drug resistant pathogens. The goal of our research is to discover a small molecular peptide that can disrupt the synthesis of new ribosomes. Using the phage display technique, we have discovered a 7-mer peptide that binds to the second strand of 16S h34 RNA with a dissociation constant in the low micromolar range. Binding of the peptide alters RNA structure and inhibits the binding of the ribosomal RNA small subunit methyltransferase C (RsmC) enzyme that methylates the exocyclic amine of G1207. The addition of this peptide also increases the lag phase of bacterial growth. Introduction of chemical modifications to increase the binding affinity of the peptide to RNA, its uptake and stability can further improve the efficacy of the peptide as an antibiotic agent against pathogenic bacteria.