RNA polymerases as moving barriers to condensin loop extrusion

“, trapped at transcription start sites by rifampicin) are directional barriers to condensin translocation, akin to CTCFs as directional barriers to cohesin in eukaryotes (4), or an RNAPindependent mechanism generates the “baseline asymmetry” of chromosome juxtaposition at loci outside of rRNA operons.”

“One can hypothesize that RNAP has a directional effect on condensin translocation, like the effect of CTCF proteins on cohesin in eukaryotes (4).”

“…do not have coupled transcription and translation, there is transcription-coupled splicing, and the spliceosome is of similar size to the ribosome (58), making the whole complex of a globular diameter > 70 nm. Future experiments will be needed to test whether SMCs can bypass steric barriers…”

“Rudner [2], Leonid A.”

“tclockwise(i) = tclockwise,operon(i) + tclockwise,rRNA(i) + tclockwise,tRNA(i) (3)”

“4A, right), when a translocating condensin encounters a transcribing RNAP, condensin is stalled and pushed back to the transcription termination site (condensin has a very low stall force measured in vitro (10) compared to RNAP (50)).”

“Although the size of isolated eukaryotic RNAPs are comparable to their bacterial counterparts [ ∼ 10 – 15 nm in diameter (56)], sizes of whole elongating transcription complexes in eukaryotes can be considerably larger.”

“Nevertheless, since SMC and CTCF interactions in eukaryotes play a regulatory role in gene expression (61), it is of interest to explore the potential role of SMC–transcription interactions in gene regulation in both bacteria and eukaryotes.”

“S8B), and there are relatively few highly transcribed genes (47); the average RNAP density is 0.”

“4 A, Right), when a translocating condensin encounters a transcribing RNAP, condensin is stalled and pushed back to the transcription termination site [condensin has a very low stall force measured in vitro (10) compared to RNAP (53)].”

“If eukaryotic RNAPs turn out as permeable to SMC as bacterial RNAPs, transcription in eukaryotes may not have a major effect on chromosome organization by loop extrusion, as compared to other molecules that specifically (and directionally) impede loop extrusion, like CTCF (20, 60).”

“4 A, Right), when a translocating condensin encounters a transcribing RNAP, condensin is stalled and pushed back to the transcription termination site [condensin has a very low stall force measured in vitro (10) compared to RNAP (53)].”

“Mirny [1,4,5]”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“Previous studies have suggested that a passive SMC ring can be pushed by transcribing RNAP (34, 50).”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“SMC complexes are characterized by a 3-part ring, composed of a dimer of SMC subunits, each with an ATPase domain and a long coiled-coil domain; a kleisin linker which closes the ring; and accessory proteins which bind to the linker to perform specific functions, depending on the organism (1).”

“Evidence also suggests that loop extrusion by cohesin SMCs underlies the formation of chromosomal domains during interphase (16 – 20).”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“2 ATP/cohesin/s (66, 67), whereas yeast condensins have higher rates of ∼1.”

“While the molecular details of this process are yet to be fully understood (14, 15), loop extrusion appears to be a mechanism that can explain a wealth of chromosomal phenomena in eukaryotes and bacteria.”

“While the molecular details of this process are yet to be fully understood (14, 15), loop extrusion appears to be a mechanism that can explain a wealth of chromosomal phenomena in eukaryotes and bacteria.”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“subtilis, as reported (7, 21), contacts from DNA segments close in the linear genome sequence, as in all organisms, give rise to the primary interaction diagonal, which extends from the bottom left to top right of the map (see Fig.”

“Predictions of the permeable moving-barriers mechanism can also be tested by single-molecule experiments similar to those that observed ATP-dependent condensin translocation (9) and loop extrusion (10) in vitro.”

“2 ATP/cohesin/s (66, 67), whereas yeast condensins have higher rates of ∼1.”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“Plausible mechanisms of SMC and RNAP interaction must solve the following puzzle: How can condensin’s effective speed of translocation [measured at >800 bp/s via Hi-C and ChIP-seq (8)] be so strongly attenuated (>20-fold) by RNAP transcription [which moves at 40–90 bp/s (49)], depending only on the relative orientatio…”

“This finding argues that dimerization (or oligomerization) of condensin may be required to obtain 2 distinct motor activities, consistent with previous experimental evidence and theoretical models (8, 14, 39, 40), or that a single 1-sided condensin dynamically switches directions of translocation, leading to apparent…”

“Amazingly, DNA juxtaposition extends from origin to terminus, generating a single 4-Mb “loop” (7, 21, 22).”

“1 RNAP/kb for most genes (48), in contrast with ∼10 RNAP/kb for rRNA genes (48) in our growth conditions (SI Appendix, section 4.”

“The maximum rate of condensin extrusion (vmax) based on the genome annotation model is related to the average rate of extrusion (vavg) by: vavg = vmax 1 Narm ∑Narm i=1 ηi , (12)”

“subtilis are co-oriented with replication (37), we posited that transcription could account for the tilt of the secondary diagonal by slowing down condensin translocation (Fig.”

“trapped at transcription start sites by rifampicin) are directional barriers to condensin translocation, akin to CTCFs as directional barriers to cohesin in eukaryotes (4), or an RNAP independent mechanism generates the “baseline asymmetry” of chromosome juxtaposition at loci outside of rRNA operons.”

“A secondary diagonal, which runs perpendicular to the primary, typical of many bacterial Hi-C interaction maps (7, 22, 27), indicates a symmetric juxtaposition of 2 chromosome arms about the parS sites located next to the origin of replication (ori) (Fig.”

“…maintaining DNA in a topological embrace [with mechanisms like “ inchworm, ” “ pumping, ” “ seg-ment-capture, ” and “ shackled-walker ” (9, 14, 25, 62 – 64)]; or 2) the SMC rings transiently open (65), disengage the topological embrace of the DNA, and reengage after passing the steric barrier…”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“Of particular interest, a recent preprint has shown that purified yeast condensins that extrude loops on DNA can bypass one another with short pauses of ∼7 s (59); this is similar to the time we measured for bacterial condensins to bypass elongating transcripts.”

“…using chromosome conformation capture (Hi-C) and chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) have shown that condensins are preferentially loaded onto chromosomes via ParB proteins bound at parS sites located primarily adjacent to the origin of replication (23 – 25).”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“In vivo experiments recently showed that translocation by condensin is an active process (8, 28): Condensin complexes travel processively and bidirectionally away from the parS loading site, in a manner that appears to be ATP-dependent, at speeds exceeding 800 bp/s (29).”

“5to 3-kb bacterial operon generates RNA of physical dimensions up to ∼60 × 30 × 10 nm (57).”

“As an example, recent studies provide strong evidence that transcription can affect genome structure and SMC action (31 – 36).”

“We generated a strain in which the sole copy of the β′ (beta prime) subunit of RNAP was fused to yellow fluorescent protein (YFP) and an SsrA tag (β′-YFP-SsrA) and could be conditionally targeted for degradation (46) (SI Appendix, Fig.”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“Tcounter−clockwise(j− 1) = Tcouter−clockwise(j) + fmix 2 · tclockwise(i) + (1− fmix) 2 · tcounter−clockwise(j), (11)”

“embrace [with mechanisms like “inchworm,” “pumping,” “segment-capture,” and “shackled-walker” (9, 14, 25, 62–64)]; or 2) the SMC rings transiently open (65), disengage the topological embrace of the DNA, and reengage after passing the steric barrier [closer to the “walker”/“rock-climber” models (39, 40)].”

“In a strain with a parS site close to a large cluster of rRNA operons (parS+26o), adding rifampicin to exponentially growing cells, which inhibits the transition from transcription initiation to elongation (42), results in a partial straightening out of the secondary diagonal as shown previously (8, 28) (Fig.”

“Once loaded, condensins progress away from the parS sites (8, 26) along both chromosomal arms, thus juxtaposing them (7, 21), resulting in the characteristic “X”-shaped pattern on the Hi-C maps of many bacteria (27).”

“Single-molecule studies provide support for this or a similar mechanism by demonstrating that budding yeast condensin SMCs are mechano-chemical motors that can translocate along DNA (9), extrude DNA loops (10), and actively compact DNA (11 – 13).”

“Rifampicin is a well-characterized transcription inhibitor that prevents the transition from transcription initiation to elongation, but does not prevent binding of RNAP to DNA (42); moreover, RNAP molecules that have started to synthesize RNA will continue to synthesize their RNA until they reach the transcription te…”

“In vivo experiments recently showed that translocation by condensin is an active process (8, 28): Condensin complexes travel processively and bidirectionally away from the parS loading site, in a manner that appears to be ATP-dependent, at speeds exceeding 800 bp/s (29).”

“ηi =  γ if condensin crosses a gene convergent with transcription ρ if condensin crosses an rRNA locus convergent with transcription 1 otherwise (13)”

“Mirny [1,4,5]”

“Accordingly, it remains a possibility that other DNA motifs or processes, correlated with gene orientations, influence condensin speeds (38).”

“Single-molecule studies provide support for this or a similar mechanism by demonstrating that budding yeast condensin SMCs are mechano-chemical motors that can translocate along DNA (9), extrude DNA loops (10), and actively compact DNA (11 – 13).”

“This finding argues that dimerization (or oligomerization) of condensin may be required to obtain 2 distinct motor activities, consistent with previous experimental evidence and theoretical models (8, 14, 39, 40), or that a single 1-sided condensin dynamically switches directions of translocation, leading to apparent…”

“…DNA in a topological embrace [with mechanisms like “ inchworm, ” “ pumping, ” “ seg-ment-capture, ” and “ shackled-walker ” (9, 14, 25, 62 – 64)]; or 2) the SMC rings transiently open (65), disengage the topological embrace of the DNA, and reengage after passing the steric barrier [closer…”

“We note, however, that this does not preclude the possibility that a single SMC complex performs the 2 motor activities: For instance, an anchored “1-sided” SMC extruder can alternate the anchoring site and the “DNA reeling arm,” effectively performing 2-sided extrusion with uncoupled kinetics (41); this will be a top…”

“Alongside these two major mechanisms, additional processes (pairing of homologs and sisters[80,81]), transcription[52,59,86] and affinities between specific genomic regions mediated by specific molecular complexes (polycomb[91], splicing factors[89], etc.”