Translation complex stabilization on messenger RNA and footprint profiling to study the RNA responses and dynamics of protein biosynthesis in the cells

Figures & data

Figure 1. Overview of the protein biosynthesis pathway (translation). (a) The mRNA translation cycle of eukaryotic and prokaryotic cells, depicting the peptide initiation, elongation and termination, and ribosome recycling phases of the cycle. The focus is brought to the start codon location differences during the initiation phase. For more details about the initiation mechanisms, refer to the respective reviews (e.g. Shirokikh and Preiss 2018; Hinnebusch et al. 2016; Hinnebusch 2017; Hershey et al. 2019; Sonenberg and Hinnebusch 2009; Gualerzi and Pon 2015; Rodnina 2018). (b) Steps of translation elongation cycle as ribosome ratchets through the codons of an open reading frame of mRNA and polymerizes the polypeptide. The focus is brought to the events and intermediates that can be specifically targeted and/or can have an impact on the translation complex footprint features and distribution. A taxa-indifferent elongation factor designation is used, with EF1 referring to eEF1A in eukaryotes, aEF1A in archaea and EF-Tu in bacteria, and EF2 referring to eEF2 in eukaryotes, aEF2 in archaea and EF-G in bacteria. In the center of the cycle schematic, the respective aminoacyl (A), peptidyl (P), and exit (E) tRNA localization and functional sites of the SSU and LSU are highlighted (populated with tRNAs from the cycle schematic to provide an example), and the location of the peptidyl transferase center in the LSU (PTC) is schematized.

Figure 2. Schematic of popular approaches to study protein biosynthesis. (a) Approaches that provide a high-throughput surveillance of protein and RNA content or report on single-molecule activity of translating ribosomes and poly(ribo)somes. (b) Polysome profiling as one of the most broadly used methods to infer translational involvement across mRNA, including in a high-throughput format when employed together with microarrays or RNA-sequencing.

Figure 3. Schematic of nuclease probing methods to study translation. (a) A classical ribosome “footprinting” method using ribosome-protected ribonuclease (RNase)-resistant fragment identification specific to certain mRNAs of choice. (b) Translation (ribosome) footprint profiling, whereby the ribosome-protected RNase-resistant mRNA fragments are isolated based on their association with the ribosomes and then subjected to high-throughput RNA sequencing. Sedimentation-based isolation of the ribosome-associated mRNA fragments is exemplified, although other methods are possible (e.g. Reid et al. 2015). In the ribosome profiling, a high-throughput sequencing-derived coverage of a matching intact mRNA often serves as a coverage control and as a way of estimating the level of transnational involvement (“translation efficiency”) of the respective mRNAs (top right plot; gene-wize comparison for all genes). The relative footprint frequency over the respective ORF codons is often used to infer ribosome dwell time and the kinetic landscape of the ORF (bottom right plot; transcript-specific features).

Figure 4. Examples of translation (ribosome) footprint profiling that use additional methods of targeted translation complex isolation, based on the presence of (left to right) specialized ribosomes, in trans interactions with the other translating ribosomes and their nascent peptides, tightly-packed nuclease-resistant stretches of ribosomes (e.g. most commonly, di- and tri-somes) and specific translation factors associated with the respective complexes.

Table 1. Summary of the major approaches employed to arrest or stabilize translational complexes on mRNA for downstream footprinting and footprint profiling.

Approach	Common conditions	Taxonomical limitations	Common footprint features	Fidelity limitations	Representative references
Individual stabilizing inhibitors of translation.	Short pretreatment of the cells with cell-permeable inhibitors (30 s–30 min), or cell collection and lysis into the inhibitor-containing buffer.	Dependent on the inhibitor specificity. Currently, stages of initiation prior to the start site recognition, and all termination and recycling steps cannot be readily stabilized.	Highly homogeneous and specific to the stabilized intermediate. The specific nature of stabilization aids in simplified methods of translation complex and footprint isolation and attribution. As different inhibitors target specific intermediates, some conclusions about the finer dynamics of e.g. elongation cycle can be made.	Followed form the specific nature of stabilization, very narrow range of intermediates that reflect natural complex distribution. Universally, biases linked with the augmented representation of stabilized intermediates following non-stabilized steps and permeability and active concentration build-up effects of delayed stabilization.	Ingolia et al. 2009; Lee et al. 2012; Dunn et al. 2013; Kannan et al. 2014; Lareau Liana et al. 2014; Yang et al. 2015; Shell et al. 2015; Basu and Yap 2016; Mohammad et al. 2016; Nakahigashi et al. 2016; Stewart et al. 2018; Iwasaki et al. 2019; Weaver et al. 2019; Wu et al. 2019; Chen YX et al. 2020, p. 202; Gelsinger et al. 2020; Kim et al. 2020; Steinberger et al. 2020; Saito et al. 2020a
Individual destabilizing inhibitors of translation	Longer pre-incubation with the destabilizing compound, allowing the development of a substantial difference between the naturally occurring intermediate abundances and the intermediate-suppressed distribution variant (10 min–24 h). Usually is followed up with a strong stabilizing inhibitor of translation, e.g. cycloheximide.	Relatively broad when applied to the stop codon read-through and termination complex destabilization due to the wide species compatibility of most of the aminoglycosides.	In agreement with the follow-up stabilizing translation inhibitor used, except the diminished abundance of the destabilized intermediates.	Mostly used as a proxy to study termination (and to some extent, recycling) events. As these inhibitors do not allow to capture the translational intermediates of interest directly, there is a difficulty quantitatively relating the magnitude of the effects to each other, particularly to the untreated control.	Wangen and Green 2020
Unspecific stabilization without small molecule inhibitors or covalent crosslinking	Rapid freezing and chilling, often followed by the use of a lysis buffer with elevated monovalent salt (e.g. 250 mM NaCl, 15 mM MgCl2 for eukaryotic cells and up to 3.4 M KCl and 100 mM MgCl2 for prokaryotic cells). Use of additional salts with divalent cations having strong RNA binding and translation inhibition properties, such as CaCl2. Use of broad inhibitors of energy-depended processes, such as non-hydrolyzable ATP and GTP analogs (e.g. AMP-PNP and GMP-PNP). Specific inhibitors and their combinations may also be employed post-freezing.	Except freezing/chilling, most of the nonspecific stabilization measures need to be optimized for the particular organism and material type, and are applied to the cell lysates (post-collection, post-freezing) rather than in vivo or in intact cells, due to the absence of cell entry capability of the respective reagents.	Broad representation of the translation intermediates and rich footprint diversity, generally with the omission of the less stable and/or shorter-lived translational complexes.	Information about fast-paced processes may be lost, and different complex types may respond differently to the, e.g. elevated salt conditions, including destabilization rather than preservation. For systems with mixed taxonomy or translational complex types (e.g. investigating cytoplasmic and mitochondrial or plastid translational complexes simultaneously), it may be difficult to find conditions of equally acceptable stabilization of the complexes.	Oh et al. 2011; Ingolia et al. 2013; Stadler and Fire 2013; Subramaniam et al. 2014; Vasquez et al. 2014; Hussmann et al. 2015; Couvillion and Churchman 2017; Shamimuzzaman and Vodkin 2018; Ivanov et al. 2018; Mohammad and Buskirk 2019; Chen YX et al. 2020; de Lomana et al. 2020
Stabilization via covalent crosslinking	Fixation with 0.025–4% w/v formaldehyde for 5 min–1 h, often under chilling on ice, and usually with reaction termination by adding, e.g. glycine or tris(hydroxymethyl)aminomethane. Other crosslinkers and inhibitors can be employed following or preceding the main fixation reaction.	The limitations mostly associate with a necessity to optimize fixative concentration and the fixation regime for the biological material with different permeability and accessibility to the crosslinker, including some fine differences in closely related types of material (e.g. mammalian cells of different type).	Maximal diversity (as well as complexity) of the footprint profile, with footprints associated with all phases of translation well-preserved and identified.	Difficulty analyzing the footprint profile due to its complexity, and as a result often complicated attribution of the footprint types to the respective original translational intermediates. Possible risks of under-crosslinking (and the depletion of short-lived intermediates) or over-crosslinking (and the depletion of heavier complexes or those in vicinity of larger intracellular structures).	Archer et al. 2016; Shirokikh et al. 2017; Bohlen, Fenzl, et al. 2020; Giess et al. 2020; Wagner et al. 2020

Figure 5. Popular specific inhibitors of translation elongation phase used to stabilize translational complexes in the footprint profiling research of eukaryotic cells. (a) Specific translation elongation inhibitors often used in translation footprint profiling research, their chemical structures and schematized mechanism of action (refer to the Figure 1 for more information on the elongation cycle context of the stabilized step). 3D (when available) and 2D structures of inhibitors were downloaded from PubChem (unless otherwise indicated). (b) Typical features of translation footprint patterns associated with the use of respective inhibitors (as indicated by arrows). Shown are schematized commonly observed variants of metagene or transcript-wise footprint distributions, aligned by footprint 5′ or 3′ ends across the sequence of a coding region of mRNA near the respective start (left, green) or stop (right, red) codons (see color version of this figure at www.tandfonline.com/ibmg).

Table 2. Examples of antibiotics and inhibitors used in ribosomal footprinting research to study peptide elongation phase of protein biosynthesis.

Antibiotic	Molecular weight, Da	Binding site	Mechanism	Specificity	Useful concentration	Footprint size, nt	Profiling studies	Other references
Footprinting elongating ribosomes
Cycloheximide	281.35	At the E-site of the LSU	Translocation inhibitor	Broad eukaryotic	10–8000 µg/ml; 35.5–3554.3 µM; Kd 0.14–15 µM	27–32, modal 28	E.g. Ingolia et al. 2009; Juntawong et al. 2014; Rubio et al. 2014; Lareau Liana et al. 2014; Yang et al. 2015; Sako et al. 2016; Sendoel et al. 2017; Stewart et al. 2018; Wu et al. 2019	E.g. Schneider-Poetsch et al. 2010; Garreau de Loubresse et al. 2014; Gerashchenko and Gladyshev 2014; Duncan and Mata 2017; Santos et al. 2019; Sharma et al. 2019
Anisomycin	265.31	At the A-site, near PTC of the LSU	Transpeptidation inhibitor	Eukaryotic, archaeal	>1 mM; Kd 10–100 µM	20–23, modal 21; 27 archaeal (broad range 15–40)	Lareau Liana et al. 2014; Wu et al. 2019; Gelsinger et al. 2020	Barbacid and Vazquez 1974; Blaha et al. 2008; Bulkley et al. 2010
Tigecycline	585.65	At the SSU A-site	Blocks accommodation of the incoming aa-tRNA in the A-site; tetracycline-like	Broad bacterial, eukaryotic	>100 μg/ml	20–23, modal 21 (assumed)	Wu et al. 2019	Jenner et al. 2013
Emetine	480.65	At the E-site of the SSU	Translocation inhibitor; pactamycin-like	Eukaryotic	20 μg/ml	30–34	Ingolia et al. 2011; Dunn et al. 2013	Wong et al. 2014
Chloramphenicol	323.13	At the A-site, near PTC of the LSU	Blocks accommodation of the incoming aa-tRNA in the A-site	Broad bacterial, mitochondrial and chloroplast	5–100 μg/ml; 1 mM	15–45	Shell et al. 2015; Basu and Yap 2016; Mohammad et al. 2019; Chen YX et al. 2020	Long and Porse 2003; Bulkley et al. 2010; Schwarz et al. 2016; Tereshchenkov et al. 2018
Thiostrepton	1664.83	Across A, P, E-sites of the PTC of the LSU	Inhibits accommodation of the incoming EF-Tu:aa-tRNA in the A-site, inhibits EF-G:GTP binding and GTP hydrolysis	Bacterial; Gram-positive	20 µM	20–40	Jeong et al. 2016; Hwang et al. 2019; Kim et al. 2020	Jonker et al. 2011; Polikanov et al. 2018
Linezolid	337.35	At the A-site, near PTC of the LSU	Blocks accommodation of the incoming aa-tRNA in the A-site	Bacterial (mostly Gram-positive)	8–800 μg/ml	15–45 (assumed)	Marks et al. 2016	Belousoff et al. 2017; Scaiola et al. 2019
Erythromycin	733.94	Near PTC of the LSU	Translocation inhibitor; macrolide family	Bacterial (mostly Gram-positive)	0.01–100 µg/ml	24–40 (assumed)	Kannan et al. 2014	Garza-Ramos et al. 2001; Borovinskaya et al. 2007; Bulkley et al. 2010; Dunkle et al. 2010; Kannan et al. 2012; Fujii et al. 2013; Jelić and Antolović 2016
Telithromycin	812.02	Near PTC of the LSU	Translocation inhibitor; macrolide family	Bacterial (mostly Gram-positive)	0.01–50 µg/ml, 1–10 µM; Kd ∼ 8 nM	24–40 (assumed)	Kannan et al. 2014	Garza-Ramos et al. 2001; Bulkley et al. 2010; Dunkle et al. 2010; Jelić and Antolović 2016
Azithromycin	748.00	Near PTC of the LSU (assumed)	Translocation inhibitor; macrolide family	Bacterial (mostly Gram-positive); apicoplasts	0.5–200 µg/ml	24–40	Davis et al. 2014	Jelić and Antolović 2016
GMP-PNP	522.20	GTP-depended translation factors (IF2, EF1, EF2, RF, etc.)	Stabilization of GTP-dependent proteins on their complex targets	All ex vivo; poor cell membrane permeability	1–10 mM or sufficient to overcome endogenous GTP and ATP	28 eukaryotic, 24–40 prokaryotic (assumed)	Li et al. 2012; Chew et al. 2013	Wilson et al. 2000; Colón-Ramos et al. 2006; Shirokikh et al. 2010

Figure 6. Same as Figure 5, but for translation elongation inhibitors commonly used in research of bacterial translation. Erythromycin structure (Fujii et al. 2013) was visualized using CCDC JSmol viewer.

Table 3. Examples of antibiotics and inhibitors used in ribosomal footprinting research to study peptide initiation phase of protein biosynthesis.

Antibiotic	Molecular weight, Da	Binding site	Mechanism	Specificity	Useful concentration	Footprint size, nt	Profiling studies	Other references
Footprinting initiating ribosomes
Harringtonin	531.60	At the A-site, near PTC of the LSU	Inhibits transpeptidation during first peptide bond formation. Does not stably bind in the presence of nascent peptide	Eukaryotic, archaeal	2 μg/ml; 1 mg/ml archaea	Archaeal 27 (also 15–19 secondary mode)	Yang et al. 2015; Stewart et al. 2018; Gelsinger et al. 2020	Fresno et al. 1977; Garreau de Loubresse et al. 2014
Lactimidomycin	457.56	At the E-site of the LSU	Inhibits translocation but cannot displace tRNA from E-site effectively, peptide elongating ribosomes can be removed with puromycin	Mostly eukaryotic	50 μM	26–29	Lee et al. 2012; Yang et al. 2015	Schneider-Poetsch et al. 2010; Garreau de Loubresse et al. 2014; Carocci and Yang 2016
Hippuristanol	462.66	RNA-depended ATPase eIF4A or eIF4A in its complex eIF4F or eIF4A:eIF4B	Binds and stabilizes eIF4A in “closed” conformation while preventing its interaction with mRNA. Does not affect ATP binding	Eukaryotic	0.05–50 μM; Kd ∼ 2 µM	Broad 25–40 range with 32 modal; cycloheximide was also used	Steinberger et al. 2020	Higa et al. 1981; Sun et al. 2014; Cencic and Pelletier 2016; Naineni et al. 2020
Rocaglamide	505.56	ssRNA:eIF4A:ATP and ssRNA:DDX3:ATP	Binds to and stabilizes eIF4A or DDX3 in “closed” conformation as it is bound to ATP and RNA; has elevated affinity to polypurine stretches. Prevents ATP hydrolysis	Eukaryotic	0.1–10 μM; Kd ∼ 0.15 eIF4A1, 0.14 eIF4A2, 0.17 DDX3 µM (with AMP-PNP)	36 median increasing up to 45	Iwasaki et al. 2016; Iwasaki et al. 2019; Chen M et al. 2020	Sadlish et al. 2013
Pateamine A	555.80	eIF4A:eIF4B	Binds to eIF4A and stabilizes eIF4A:eIF4B and induces ATPase while destabilizing eIF4G:eIF4A binding	Eukaryotic	1–10 μM	26–36; −14 footprint 5′ end	Popa et al. 2016	Low et al. 2005; Khong et al. 2016
Pactamycin	558.60	At the E-site of the SSU	Displaces mRNA in the P- and E-sites; likely interferes with E-site tRNA	Broad	5 μg/ml	25–50	Nakahigashi et al. 2016	Brodersen et al. 2000; Lu et al. 2011
Tetracycline	444.44	Near A-site of the SSU; other binding sites in SSU and LSU (non-essential)	Competitively ejects aa-tRNA after GTP hydrolysis (preventing transpeptidation)	Broad bacterial, mitochondrial	12–40 μg/ml	25–50	Nakahigashi et al. 2016; Hwang and Buskirk 2017	Brodersen et al. 2000; Pioletti et al. 2001; Wilson 2014; Dmitriev et al. 2020
Clindamycin	424.98	At the A-site of the LSU, near PTC, near peptide tunnel entry	Interferes with the aa-tRNA binding	Bacterial (mostly Gram-positive)	10–100 μg/ml; Kd ∼ 450 μM; IC50 0.6 μg/ml E. coli, 0.07 μg/ml S. aureus	25–50	Nakahigashi et al. 2016	Tu et al. 2005; Kouvela et al. 2006; Dunkle et al. 2010; Garreau de Loubresse et al. 2014; Matzov et al. 2017
Retapamulin	517.77	Near P-site of the LSU, mostly at the A-site and covering some of the P-site	Transpeptidation inhibitor; pleuromutilin derivative	Bacteria (mostly Gram-positive)	5–15 μg/ml; Kd ∼ 3 nM	25–45; +14–16 footprint 3′ end	Meydan et al. 2019; Weaver et al. 2019; Saito et al. 2020a	Poulsen et al. 2001; Schlünzen et al. 2004; Yan et al. 2006; Davidovich et al. 2007
Oncocin (Onc112)	2,389.80	At the PTC of the LSU, mostly near A-site and covering peptide exit tunnel	Multifunctional elongation inhibition by blocking A-site and partially P-site of the LSU, PTC and peptide exit; H-VDKPPYLPRPRPPRrIYNr-Amidation	Broad bacterial	1–100 μM; Kd ∼ 90 nM	+6–10 footprint 3′ end	Weaver et al. 2019	Krizsan et al. 2014; Mardirossian et al. 2014, p. 7; Lomakin et al. 2015; Roy et al. 2015, p. 112; Seefeldt et al. 2015; Arenz and Wilson 2016
Lincomycin	406.54	At the A-site of the LSU, near PTC, near peptide tunnel entry	Translocation inhibitor; linkosamide, macrolide-like	Bacterial (mostly Gram-positive), plastids/ mitochondria	IC50 1.7 μg/ml E. coli, 0.03 μg/ml S. aureus	20–40; library clipping	Chotewutmontri and Barkan 2018; Fujita et al. 2019	Dunkle et al. 2010; Matzov et al. 2017
AMP-PNP	529.93	Binds to ATP- and RNA-dependent RNA helicases and RNA binding proteins	Inhibits β-γ ATPases (adenosine triphosphate monophosphohydrolases)	All ex vivo; poor cell membrane permeability	1–10 mM or sufficient to displace endogenous ATP	34; broad range extending to >45	Iwasaki et al. 2016	Pestova and Kolupaeva 2002; Shirokikh and Spirin 2008; Shirokikh et al. 2019

Figure 7. Same as Figure 5, but for translation inhibitors and their combinations commonly used to study eukaryotic translation initiation mechanisms.

Figure 8. Small compounds targeting eukaryotic translation initiation factor and ATP-dependent single-stranded RNA binding protein (RNA helicase) eIF4A and its interactions with the substrates (mRNA, ATP) and other initiation factors and complexes, such as eIF4G:SSU and eIF4B. For more details about scanning and eIF4A action, refer to the specialized reviews (e.g. Shirokikh and Preiss 2018; Hinnebusch et al. 2016; Hinnebusch 2017; Naineni et al. 2020). Schematized mechanism of action of each compound is depicted. Note that in the majority of the observations, the footprint signal is found to be concentrated at or nearby the respective ORF start sites, and is observed in the footprints derived from the fraction of translational complexes containing complete ribosomes (as opposed to SSUs).

Figure 9. Same as Figure 7, but for translation inhibitors commonly used to study bacterial translation initiation mechanisms. Oncocin Onc112 PDB structure 4ZER was visualized using PDB Mol viewer (Seefeldt et al. 2015).

Figure 10. Specific translation termination inhibitors often used in translation footprint profiling research. (a) Mechanism of action and the stabilized state of the ribosome characteristic to the exemplified compounds. Note that the currently used translation termination inhibitors are as such inducers of read-through and thus, unlike inhibitors used to study the other phases of translation, in fact de-stabilize translation termination complexes. The shown compounds have a broad specificity across taxa and are used in both, eukaryotic and bacterial research. (b) Typical meatgene features of translation footprint patterns associated with the use of respective inhibitors (for designations, refer to Figure 5(b)) using an eukaryotic system as example.

Table 4. Examples of antibiotics and inhibitors used in ribosomal footprinting research to study peptide termination and ribosomal recycling phases of protein biosynthesis.

Antibiotic	Molecular weight, Da	Binding site	Mechanism	Specificity	Useful concentration	Footprint size, nt	Profiling studies	Other references
Footprinting termination and recycling
Geneticin (G418)	496.55	Decoding center; mostly near SSU A-site	Inhibit inersubunit rotation, cause miscoding by interference and conformational alterations at the A-site	Broad	5–2000 μg/ml;	30–31 (eukaryotic)	Wangen and Green 2020	Eustice and Wilhelm 1984; Krause et al. 2016; Prokhorova et al. 2017
Gentamicin	477.60							Borovinskaya et al. 2007; Krause et al. 2016; Prokhorova et al. 2017
Paromomycin	615.63							Fourmy et al. 1996; Arenz and Wilson 2016; Krause et al. 2016; Prokhorova et al. 2017
Neomycin	614.65							Borovinskaya et al. 2007; Krause et al. 2016
Tobramycin	467.52							Salian et al. 2012; Krause et al. 2016
Amikacin	585.60							Krause et al. 2016; Ramirez and Tolmasky 2017

Figure 11. Combinations of specific translation inhibitors and their effects to study the dynamics of translation elongation and translation complex identity, as well as codon specificity, of the stalled intermediates in eukaryotic systems. (a) Cycloheximide chase following harringtonin translation initiation arrest to infer the rate of ribosomal progression along mRNA ORFs. Note that the translation initiation is stopped upon the completion of the start codon recognition and the assembly of the elongation-capable ribosome at the respective start sites. (b) Use of tigecycline-cycloheximide and anisomycin-cycloheximide combinations to arrest ribosomes with differently configured A-sites, populated (left, bottom; blocked transpeptidation) and non-populated (right, top; blocked aminoacyl-tRNA accommodation) with the incoming aminoacyl-tRNA:eEF1A:GTP, respectively. Shorter footprints (left, bottom) in this case are reflective of the delays in attracting the respective aminoacyl-tRNA, whereas longer footprints (right, top) are reflective of eEF2 deficiency (see text for more details).

Figure 12. Commonly accepted inhibitor-free approaches to preserve translational complexes on mRNA.

Figure 13. Overview of translation complex footprinting methods based on in vivo stabilization of translation intermediates with covalent crosslinking. (a) Example of a possible formaldehyde-mediated RNA-protein crosslink formation. (b) Schematized TCP-seq, RCP-seq, 40S ribosome profiling and Sel-TCP-seq methods (see text for more details). Characteristic footprint patterns and features for both, SSU- and ribosome-derived footprints are shown, covering the entire translation cycle on mRNA. (c) Factor-selective methods using covalent stabilization allow to obtain additional insight into translation factor involvement along the translation cycle phases (left), and refine start selection mechanisms and dynamics, as well as detect and monitor co-translational complex assembly (right). Designations and structures as in Figure 5.

Figure 14. Formaldehyde-based covalent stabilization of translational complexes is a versatile approach compatible with rapid protein biosynthesis dynamics. (a) Estimates of formaldehyde vs. cycloheximide characteristic diffusion times to a given % of their initial concentration (blue line demarks 90%) over a typical barrier (5 µm for attached cell thickness or 50 µm for a group of cells or tissue fragment). Diffusion coefficients in water and a 2-term Taylor series approximation of the Fick’s law were used; calculations and plotting was done in GeoGebra (https://www.geogebra.org). (b,c) Mapping of HEK 293 formaldehyde-fixed TCP-seq-style footprints demonstrating the presence of footprints from nuclease-resistant disome fraction (b), specific signals from mitochondrial SSU and ribosomes in the respective fractions, and mitochondrial gene coverage in all, SSU, ribosome and disome fractions (c). These results suggest universal and taxonomically indifferent applicability of the formaldehyde-based covalent stabilization of translational complexes (see color version of this figure at www.tandfonline.com/ibmg).

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