Publications by Nicole Robb


Single-Molecule Analysis of the Influenza Virus Replication Initiation Mechanism

BIOPHYSICAL JOURNAL 114 (2018) 246A-246A

NC Robb, AJW te Velthuis, E Fodor, AN Kapanidis


Coming together during viral assembly.

Nature reviews. Microbiology 16 (2018) 721-721

C Hepp, NC Robb


Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Nature methods 15 (2018) 669-676

B Hellenkamp, S Schmid, O Doroshenko, O Opanasyuk, R Kühnemuth, S Rezaei Adariani, B Ambrose, M Aznauryan, A Barth, V Birkedal, ME Bowen, H Chen, T Cordes, T Eilert, C Fijen, C Gebhardt, M Götz, G Gouridis, E Gratton, T Ha, P Hao, CA Hanke, A Hartmann, J Hendrix, LL Hildebrandt, V Hirschfeld, J Hohlbein, B Hua, CG Hübner, E Kallis, AN Kapanidis, J-Y Kim, G Krainer, DC Lamb, NK Lee, EA Lemke, B Levesque, M Levitus, JJ McCann, N Naredi-Rainer, D Nettels, T Ngo, R Qiu, NC Robb, C Röcker, H Sanabria, M Schlierf, T Schröder, B Schuler, H Seidel, L Streit, J Thurn, P Tinnefeld, S Tyagi, N Vandenberk, AM Vera, KR Weninger, B Wünsch, IS Yanez-Orozco, J Michaelis, CAM Seidel, TD Craggs, T Hugel

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.


Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.

Nucleic acids research 46 (2018) 677-688

D Duchi, K Gryte, NC Robb, Z Morichaud, C Sheppard, K Brodolin, S Wigneshweraraj, AN Kapanidis

Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP-DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.


Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Nature methods 15 (2018) 984-984

B Hellenkamp, S Schmid, O Doroshenko, O Opanasyuk, R Kühnemuth, SR Adariani, B Ambrose, M Aznauryan, A Barth, V Birkedal, ME Bowen, H Chen, T Cordes, T Eilert, C Fijen, C Gebhardt, M Götz, G Gouridis, E Gratton, T Ha, P Hao, CA Hanke, A Hartmann, J Hendrix, LL Hildebrandt, V Hirschfeld, J Hohlbein, B Hua, CG Hübner, E Kallis, AN Kapanidis, J-Y Kim, G Krainer, DC Lamb, NK Lee, EA Lemke, B Levesque, M Levitus, JJ McCann, N Naredi-Rainer, D Nettels, T Ngo, R Qiu, NC Robb, C Röcker, H Sanabria, M Schlierf, T Schröder, B Schuler, H Seidel, L Streit, J Thurn, P Tinnefeld, S Tyagi, N Vandenberk, AM Vera, KR Weninger, B Wünsch, IS Yanez-Orozco, J Michaelis, CAM Seidel, TD Craggs, T Hugel

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.


The role of the priming loop in Influenza A virus RNA synthesis.

Nature microbiology 1 (2016)

AJW Te Velthuis, NC Robb, AN Kapanidis, E Fodor

RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes1. They adopt a closed, right-handed fold with conserved subdomains called palm, fingers, and thumb1,2. Conserved RdRp motifs A-F coordinate the viral RNA template, NTPs, and magnesium ions to facilitate nucleotide condensation1. For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism3. The Influenza A virus RdRp in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication4. To understand how the Influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation, and single-molecule FRET assays. Our data shows that this β-hairpin is essential for terminal initiation during replication, but auxiliary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of Influenza A virus RNA synthesis and identifies the initiation platform of viral replication.


Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA.

Nucleic acids research 44 (2016) 10304-10315

NC Robb, AJW Te Velthuis, R Wieneke, R Tampé, T Cordes, E Fodor, AN Kapanidis

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.


The role of the priming loop in influenza A virus RNA synthesis.

Nature microbiology 1 (2016) 16029-

AJW Te Velthuis, NC Robb, AN Kapanidis, E Fodor

RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes(1). They adopt a closed, right-handed fold with conserved subdomains called palm, fingers and thumb(1,2). Conserved RdRp motifs A-F coordinate the viral RNA template, NTPs and magnesium ions to facilitate nucleotide condensation(1). For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism(3). The influenza A virus RdRp, in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication(4). To understand how the influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation and single-molecule Förster resonance energy transfer (sm-FRET) assays. Our data indicate that this β-hairpin is essential for terminal initiation during replication, but not necessary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of influenza A virus RNA synthesis and identifies the initiation platform of viral replication.


RNA Polymerase Pausing during Initial Transcription.

Molecular cell 63 (2016) 939-950

D Duchi, DLV Bauer, L Fernandez, G Evans, N Robb, LC Hwang, K Gryte, A Tomescu, P Zawadzki, Z Morichaud, K Brodolin, AN Kapanidis

In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.


Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter.

Proceedings of the National Academy of Sciences of the United States of America 111 (2014) E3335-E3342

AI Tomescu, NC Robb, N Hengrung, E Fodor, AN Kapanidis

The influenza virus is a major human and animal pathogen responsible for seasonal epidemics and occasional pandemics. The genome of the influenza A virus comprises eight segments of single-stranded, negative-sense RNA with highly conserved 5' and 3' termini. These termini interact to form a double-stranded promoter structure that is recognized and bound by the viral RNA-dependent RNA polymerase (RNAP); however, no 3D structural information for the influenza polymerase-bound promoter exists. Functional studies have led to the proposal of several 2D models for the secondary structure of the bound promoter, including a corkscrew model in which the 5' and 3' termini form short hairpins. We have taken advantage of an insect-cell system to prepare large amounts of active recombinant influenza virus RNAP, and used this to develop a highly sensitive single-molecule FRET assay to measure distances between fluorescent dyes located on the promoter and map its structure both with and without the polymerase bound. These advances enabled the direct analysis of the influenza promoter structure in complex with the viral RNAP, and provided 3D structural information that is in agreement with the corkscrew model for the influenza virus promoter RNA. Our data provide insights into the mechanisms of promoter binding by the influenza RNAP and have implications for the understanding of the regulatory mechanisms involved in the transcription of viral genes and replication of the viral RNA genome. In addition, the simplicity of this system should translate readily to the study of any virus polymerase-promoter interaction.


Characterizing Influenza a RNA Polymerase - Promoter Interaction using Ensemble Fluorescence Spectroscopy

BIOPHYSICAL JOURNAL 104 (2013) 584A-584A

AI Tomescu, NC Robb, N Hengrung, E Fodor, AN Kapanidis


The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection.

J Mol Biol 425 (2013) 875-885

NC Robb, T Cordes, LC Hwang, K Gryte, D Duchi, TD Craggs, Y Santoso, S Weiss, RH Ebright, AN Kapanidis

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labeling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.


The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: Implications for transcription start-site selection

Journal of Molecular Biology 425 (2013) 875-885

NC Robb, T Cordes, LC Hwang, K Gryte, D Duchi, TD Craggs, Y Santoso, S Weiss, RH Ebright, AN Kapanidis

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~ 14 bp around the transcription start site and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RPo). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5′ end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo. The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RPo with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo using different FRET rulers and labeling positions. An analysis of FRET distributions of RP o using burst variance analysis reveals conformational fluctuations in RPo in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RPo. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RPo and indicates that DNA dynamics within the bubble affect the search for transcription start sites. ©2013 Elsevier Ltd. All rights reserved.


The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein

Journal of General Virology 93 (2012) 113-118

NC Robb, E Fodor

The influenza A virus M1 mRNA is alternatively spliced to produce M2 mRNA, mRNA 3, and in some cases, M4 mRNA. Splicing of influenza mRNAs is carried out by the cellular splicing machinery and is thought to be regulated, as both spliced and unspliced mRNAs encode proteins. In this study, we used radioactively labelled primers to investigate the accumulation of spliced and unspliced M segment mRNAs in viral infection and ribonucleoprotein (RNP) reconstitution assays in which only the minimal components required for transcription and replication to occur were expressed. We found that co-expression of the viral NS1 protein in an RNP reconstitution assay altered the accumulation of spliced mRNAs compared with when it was absent, and that this activity was dependent on the RNA-binding ability of NS1. These findings suggest that the NS1 protein plays a role in the regulation of splicing of influenza virus M1 mRNA. © 2012 SGM.


The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein.

The Journal of general virology 93 (2012) 113-118

NC Robb, E Fodor

The influenza A virus M1 mRNA is alternatively spliced to produce M2 mRNA, mRNA(3), and in some cases, M4 mRNA. Splicing of influenza mRNAs is carried out by the cellular splicing machinery and is thought to be regulated, as both spliced and unspliced mRNAs encode proteins. In this study, we used radioactively labelled primers to investigate the accumulation of spliced and unspliced M segment mRNAs in viral infection and ribonucleoprotein (RNP) reconstitution assays in which only the minimal components required for transcription and replication to occur were expressed. We found that co-expression of the viral NS1 protein in an RNP reconstitution assay altered the accumulation of spliced mRNAs compared with when it was absent, and that this activity was dependent on the RNA-binding ability of NS1. These findings suggest that the NS1 protein plays a role in the regulation of splicing of influenza virus M1 mRNA.


Single-Molecule Studies of Transcription Initiation using a Vesicle Approach

BIOPHYSICAL JOURNAL 102 (2012) 485A-485A

DD Llumigusin, N Robb, K Gryte, AN Kapanidis


Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

PLoS Pathog 8 (2012) e1002998-

HM Wise, EC Hutchinson, BW Jagger, AD Stuart, ZH Kang, N Robb, LM Schwartzman, JC Kash, E Fodor, AE Firth, JR Gog, JK Taubenberger, P Digard

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.


The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes

Journal of Virology 85 (2011) 5228-5231

NC Robb, G Chase, K Bier, FT Vreede, PC Shaw, N Naffakh, M Schwemmle, E Fodor

The influenza A virus genome consists of eight RNA segments that associate with the viral polymerase proteins (PB1, PB2, and PA) and nucleoprotein (NP) to form ribonucleoprotein complexes (RNPs). The viral NS1 protein was previously shown to associate with these complexes, although it was not clear which RNP component mediated the interaction. Using individual TAP (tandem affinity purification)-tagged PB1, PB2, PA, and NP, we demonstrated that the NS1 protein interacts specifically with NP and not the polymerase subunits. The region of NS1 that binds NP was mapped to the RNA-binding domain. © 2011, American Society for Microbiology.


The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes.

Journal of Virology 85 (2011) 5228-5231

NC Robb, G Chase, K Bier, FT Vreede, P-C Shaw, N Naffakh, M Schwemmle, E Fodor

The influenza A virus genome consists of eight RNA segments that associate with the viral polymerase proteins (PB1, PB2, and PA) and nucleoprotein (NP) to form ribonucleoprotein complexes (RNPs). The viral NS1 protein was previously shown to associate with these complexes, although it was not clear which RNP component mediated the interaction. Using individual TAP (tandem affinity purification)-tagged PB1, PB2, PA, and NP, we demonstrated that the NS1 protein interacts specifically with NP and not the polymerase subunits. The region of NS1 that binds NP was mapped to the RNA-binding domain.


Functional analysis of the influenza virus H5N1 nucleoprotein tail loop reveals amino acids that are crucial for oligomerization and ribonucleoprotein activities

Journal of Virology 84 (2010) 7337-7345

WH Chan, AKL Ng, NC Robb, MKH Lam, PKS Chan, SWN Au, JH Wang, E Fodor, PC Shaw

Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

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