Furqan M. Fazal

Real-time observation of the initiation of RNA polymerase II transcription.

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a ‘closed’ preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an ‘open’ complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

Circular differential double diffraction in chiral media

In an optically active liquid the diffraction angle depends on the circular polarization state of the incident light beam. We report the observation of circular differential diffraction in an isotropic chiral medium, and we demonstrate that double diffraction is an alternate means to determine the handedness (enantiomeric excess) of a solution.

Direct observation of processive exoribonuclease motion using optical tweezers

Significance Bacteria regulate the synthesis and degradation of RNA molecules to ensure timely and robust responses to an ever-changing environment. The transcript’s lifetime can be influenced profoundly by a secondary structure that can form in the RNA and that may inhibit or promote its digestion by RNases. The molecular mechanisms by which RNases interact with structured RNAs therefore are of great interest. In this study we used optical tweezers to investigate the mechanistic properties of two such enzymes from Escherichia coli, polynucleotide phosphorylase and RNase R. Our results offer new insights into the functional characteristics of these two enzymes, including the sequence-dependent behavior of RNase R and the presence of discrete steps of six or seven nucleotides taken by polynucleotide phosphorylase. Bacterial RNases catalyze the turnover of RNA and are essential for gene expression and quality surveillance of transcripts. In Escherichia coli, the exoribonucleases RNase R and polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor the translocation of individual enzymes along RNA-based substrates. Single-molecule records of motion reveal RNase R to be highly processive: one molecule can unwind over 500 bp of a structured substrate. However, enzyme progress is interrupted by pausing and stalling events that can slow degradation in a sequence-dependent fashion. We found that the distance traveled by PNPase through structured RNA is dependent on the A+U content of the substrate and that removal of its KH and S1 RNA-binding domains can reduce enzyme processivity without affecting the velocity. By a periodogram analysis of single-molecule records, we establish that PNPase takes discrete steps of six or seven nucleotides. These findings, in combination with previous structural and biochemical data, support an asymmetric inchworm mechanism for PNPase motion. The assay developed here for RNase R and PNPase is well suited to studies of other exonucleases and helicases.

Spectral Energy Distributions of High-Mass Protostellar Objects: Evidence of High Accretion Rates

The spectral energy distributions (SEDs), spanning the mid-infrared to millimeter wavelengths, of a sample of 13 high-mass protostellar objects (HMPOs) were studied using a large archive of 2D axisymmetric radiative transfer models. Measurements from the Spitzer GLIMPSE and MIPSGAL surveys and the MSX survey were used in addition to our own surveys at millimeter and submillimeter wavelengths to construct the SEDs, which were then fit to the archive of models. These models assumed that stars of all masses form via accretion and allowed us to make estimates for the masses, luminosities, and envelope accretion rates for the HMPOs. The models fit the observed SEDs well. The implied envelope accretion rates are high, ≈10−2.5 M☉ yr−1, consistent with the accretion-based scenario of massive star formation. With the fitted accretion rates and with mass estimates of up to ~20 M☉ for these objects, it appears plausible that stars with stellar masses M* > 20 M☉ can form via accretion.

Atlas of Subcellular RNA Localization Revealed by APEX-seq

We introduce APEX-seq, a method for RNA sequencing based on spatial proximity to the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome, revealing extensive and exquisite patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

Dynamic Regulation of RNA Structure in Mammalian Cells

RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, termed RNA structuromes. However, previous whole-cell analyses lacked the resolution to unravel the dynamic regulation of RNA structure across subcellular states. Here we reveal the RNA structuromes in three compartments — chromatin, nucleoplasm and cytoplasm. The cytotopic structuromes substantially expand RNA structural information, and enable detailed investigation of the central role of RNA structure in linking transcription, translation, and RNA decay. Through comparative structure analysis, we develop a resource to visualize the interplay of RNA-protein interactions, RNA chemical modifications, and RNA structure, and predict both direct and indirect reader proteins of RNA modifications. We validate the novel role of the RNA binding protein LIN28A as an N6-methyladenosine (m6A) modification “anti-reader”. Our results highlight the dynamic nature of RNA structures and its functional significance in gene regulation.

Real-time observation of polymerase-promoter contact remodeling during transcription initiation

Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate not only the strength of promoter binding, but also the frequency and timing of promoter escape during transcription. Here, we describe a single-molecule optical-trapping assay to study transcription initiation in real time, and use it to map contacts formed between σ70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to observe the remodeling of those contacts during the transition to the elongation phase. The strong binding contacts identified in certain well-known promoter regions, such as the −35 and −10 elements, do not necessarily coincide with the most highly conserved portions of these sequences. Strong contacts formed within the spacer region (−10 to −35) and with the −10 element are essential for initiation and promoter escape, respectively, and the holoenzyme releases contacts with promoter elements in a non-sequential fashion during escape.Contacts between RNA polymerase and promoter DNA modulate the strength of binding and the frequency of promoter escape during transcription. Here, the authors describe a single molecule optical-trapping assay to study transcription initiation and observe the dynamic remodeling of enzyme contacts in real time.

Real-Time Observation of the Initiation of RNA Polymerase II Transcription

Biochemical and structural studies have shown that the initiation of RNA polymerase II (Pol II) transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a ‘closed’ preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an ‘open’ complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. We have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers (Fazal et al., Nature 2015). Contrary to expectation, scanning driven by the transcription factor IIH (TFIIH) involved the rapid opening of an extended bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.