High-throughput single-molecule optofluidic analysis

Kim, Soo-Hong; Streets, Aaron; Lin, Ron R.; Quake, Stephen R.; Weiss, Shimon; Majumdar, Devdoot

doi:10.1038/nmeth.1569

Cited by 95 publications

(114 citation statements)

References 23 publications

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“…RP O is then incubated in solution at 37°C for 30 min. To remove unreacted and nonspecifically bound RNAP, 1 μL of 100 mg/mL Heparin-Sepharose CL-6B beads (GE Healthcare) is added to RP O solution together with 10 μL of prewarmed 1× transcription buffer [Heparin challenge (3,65)]. The mixture is incubated for 1 min at 37°C and centrifuged for at least 45 s at 6,000 rpm.…”

Section: Methodsmentioning

confidence: 99%

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Lerner

Chung

Allen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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111

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Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2-or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the pausedbacktracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.T ranscription in Escherichia coli comprises three stages: initiation, elongation and termination. Initiation, which is typically the rate-limiting and the most regulated stage of transcription, is by itself a complex, multistep process consisting of the following successive steps (1, 2): (i) association of RNA polymerase (RNAP) core enzyme (subunit composition α2ββ′ω) with the promoter specificity factor σ (such as σ70 for transcription of housekeeping genes) to form RNAP holoenzyme; (ii) binding of holoenzyme to the −10 and −35 DNA elements in the promoter recognition sequence (PRS) upstream to the transcription start site (TSS) to form closed promoter complex (RPc); (iii) isomerization of RPc through multiple intermediates into an open promoter complex (RPo), in which a ∼12-bp DNA stretch (bases at registers −10 to +2) is melted to form a transcription bubble, the template DNA strand is inserted into RNAP major cleft positioning the base at register +1 of TSS at the active site, the nontemplate strand is tightly bound by σ70 and the downstream DNA duplex (bases +3 up to +20) is loaded into RNAP β′ DNA-binding clamp; (iv) an abortive initiation step (AI), where binding of nucleoside triphosphate (NTPs) and the start of RNA synthesis leads to formation of an initial transcribing complex (RP ITC ) followed by RNAP cycling through multiple polymerization trials via a DNA scrunching mechanism (3, 4), release of short "abortive transcripts" and repositioning itself in RP O for a new synthesis trial (5-7); and finally, (v) RNAP promoter escape, when enough strain is built in the enzyme, the σ70 undergoes structural transition to relieve blockage of ...

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Section: Methodsmentioning

confidence: 99%

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Lerner

Chung

Allen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

111

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“…9B also shows data points from a series of quenched kinetics experiments red square), obtained with the same system but a completely different experimental strategy (see ref. [43] for details). In this approach, a doubly-labeled complementary ssDNA probe hybridizes with the RNA transcript once the transcription reaction is stopped after a specified reaction time t. Hybridization of the probe results in a shift from high to low FRET.…”

Section: Resultsmentioning

confidence: 99%

“…The mixture was incubated for 5 min at 37 ºC and centrifuged for at least 45 s at 6,000 rpm. 20 μl of the supernatant containing RPO wass transferred into a new tube containing 10 μl of pre-warmed 1X transcription buffer (heparin challenge [43,44]). …”

Section: Multispot Fcs Analysis Resultsmentioning

confidence: 99%

Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

Ingargiola

Lerner

Chung

et al. 2016

Preprint

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We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription.Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

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“…9,14,18,[37][38][39][40][41][42][43][44] Recently, using different experimental approaches, Duchi et al 1 and Lerner, Chung et al 2 have provided direct evidence of E. coli RNAP transcription pausing during initiation at a modified lac promoter, lacCONS. 45 Lerner, Chung et al measured the kinetics of single-round transcription reactions with unlabeled reconstituted RNAP holoenzyme and promoter DNA free in solution. They observed that formation of a full-length transcript starting from a late initiation stage (e.g., initially transcribing RNAP promoter complex, ITC, synthesizing abortive transcripts of up to 7 nucleotides (nt) in length, RP ITC 7 ) was delayed as compared with transcription starting from early initiation stages (order of delays: RP ITC 7 > RP ITC 6 > RP ITC 4 > RP ITC D 2 ).…”

Section: Experimental Evidence Of Pausing During Transcription Initiamentioning

confidence: 99%

Different types of pausing modes during transcription initiation

et al. 2017

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In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape. The previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi et al. 1 and from Lerner, Chung et al. 2 In both studies, pausing is attributed to a lack of forward translocation of the nascent transcript during initiation. However, the two works report on different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we discuss these studies, together with additional experimental results from single-molecule FRET focusing on a specific distance within the transcription bubble. We show that the results of these studies are complementary to each other and are consistent with a model involving two types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer nascent transcript and a long-lived pause that occurs as a result of 1-2 nucleotide backtracking of a 7-mer transcript. Prolonged periods during which RNA polymerase (RNAP) resides in a specific state can be used for efficient control and regulation of DNA transcription. This drives us to study the slow processes that limit the overall rate of transcription.Transcription is generally divided into three major stages: initiation, elongation and termination, where initiation is usually the rate-limiting stage. Initiation can be further divided into sub-stages.3,4 First, the RNA polymerase (RNAP) holoenzyme is formed by the association of the core enzyme (subunit composition a 2 ,b,b 0 ,v) with the specificity subunit s. Different s factors recognize specific promoter sequences of various groups of genes. The major s factor in Escherichia coli, s70, is responsible for recognition of promoters for house-keeping genes. After association with the RNAP holoenzyme, s70 binds to the promoter DNA to form the RNAP promoter closed complex (RPc), which involves binding at two hexamer sequences centered around positions ¡10 and ¡35 relative to the C1 transcription start site. After binding to the promoter, RNAP forms the RNAP promoter open complex (RPo) through successive isomerization steps. 5,6 During this process around 13 bp of DNA (from registers ¡11 to C2) must melt to form the transcription bubble. The sequence of the template strand of the transcription bubble serves as a reading guide for the polymerization of an RNA transcript through base complementation. The two most downstream DNA bases in the template strand of the transcription bubble are coordinated with the active site of RNAP, where the addition of NTPs into the 3 0 end of the nascent transcript occurs. The downstream fork of the transcription b...

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High-throughput single-molecule optofluidic analysis

Cited by 95 publications

References 23 publications

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

Different types of pausing modes during transcription initiation

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