SrmB Rescues Trapped Ribosome Assembly Intermediates

Journal Pre-proof SrmB Rescues Trapped Ribosome Assembly Intermediates Jessica N. Rabuck-Gibbons, Anna M. Popova, Emily M. Greene, Carla F. Cervantes, Dmitry Lyumkis, James R. Williamson PII:

S0022-2836(19)30713-2

DOI:

https://doi.org/10.1016/j.jmb.2019.12.013

Reference:

YJMBI 66354

To appear in:

Journal of Molecular Biology

Received Date: 29 June 2019 Revised Date:

3 December 2019

Accepted Date: 4 December 2019

Please cite this article as: J.N. Rabuck-Gibbons, A.M. Popova, E.M. Greene, C.F. Cervantes, D. Lyumkis, J.R Williamson, SrmB Rescues Trapped Ribosome Assembly Intermediates, Journal of Molecular Biology, https://doi.org/10.1016/j.jmb.2019.12.013. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Binding along 5’-3’ rRNA axis

5’

3’

ΔsrmB

23S rRNA 24

20 21 4

17

3

23

1

5 18

Full Occupancy

29 13 22

Partial Occupancy

15 19

34

32

14

5S

2 11

30

35

36

7/12

28 10

16

6

9

33 27

25

R-Proteins Binding to rRNA

31

CryoEM

rRNA Modification Changes

1

SrmB

Rescues

Trapped

Ribosome

Assembly

2

Intermediates

3

Jessica N. Rabuck-Gibbonsa,b, Anna M. Popovaa, Emily M. Greenea,

4

Cervantesa, Dmitry Lyumkisb, James R. Williamsona*

5

a

6

Research Institute, 10550 N Torrey Pines Road, La Jolla, California, 92037.

7

b

8

for Biological Studies, 10010 N Torrey Pines Road, La Jolla, California 92037.

9

*

Carla F.

Department of Integrative, Structural and Computational Biology, The Scripps

Laboratory of Genetics and Helmsley Center for Genomic Medicine, The Salk Institute

To whom correspondence should be addressed: [email protected], (858) 784-8740.

10

Key Words: SrmB; DEAD-box Helicase; Ribosome Biogenesis; Cryo-electron

11

Microscopy; Quantitative Mass Spectrometry

12 13

Abstract

14

RNA helicases play various roles in ribosome biogenesis depending on the ribosome assembly

15

pathway and stress state of the cell. However, it is unclear how most RNA helicases interact

16

with ribosome assembly intermediates or on other cell processes to regulate ribosome

17

assembly. SrmB is a DEAD-box helicase that acts early in the ribosome assembly process,

18

although very little is known about its mechanism of action. Here, we use a combined

19

quantitative mass spectrometry/cryo-electron microscopy approach to detail the protein

20

inventory, rRNA modification state, and structures of 40S ribosomal intermediates that form

21

upon SrmB deletion. We show that the binding site of SrmB is unperturbed by SrmB deletion,

22

but the peptidyl transferase center, the uL7/12 stalk, and 30S contact sites all show severe

23

assembly defects. Taking into account existing data on SrmB function and the experiments

1

1

presented here, we propose several mechanisms by which SrmB could guide assembling

2

particles from kinetic traps to competent subunits during the 50S ribosome assembly process.

3 4

Introduction

5

The ribosome is a large dynamic ribonucleoprotein (RNP) machine responsible for protein

6

synthesis, a key process for all organisms. Disruption of protein synthesis leads to well-

7

documented defects in cell growth and cell cycle control that are, in turn, translated to various

8

disease states[1]. In Escherichia coli (E. coli), the 70S ribosome is composed of 54 ribosomal

9

proteins (r-proteins) and three ribosomal RNAs (rRNAs) assembled into two subunits: the small

10

(30S) and large (50S) subunits. The 50S large subunit is formed by association of the 23S and

11

5S rRNAs and 33 proteins (bL1 to bL36) (Fig. 1), and its functions include catalysis of the

12

peptidyl transfer reaction and translocation along mRNA, preventing premature nascent chain

13

hydrolysis, providing the binding site for tRNA and factors that assist in initiation, elongation and

14

termination, and facilitation of protein folding after synthesis[2-5] While the functional states of

15

the translating ribosome have been well characterized, comparatively little of the ribosome

16

assembly process is understood.

17 18

RNA helicases are an important class of ribosome assembly factors, and the majority of RNA

19

helicases found in E. coli are involved in ribosome biogenesis[6]. In particular, the DEAD-box

20

family of proteins contains RNA helicases that are critically involved in nearly all aspects of

21

cellular RNA metabolism[6]. The DEAD-box helicases are characterized by a highly conserved

22

helicase core, containing at least 12 conserved amino acid motifs that participate in binding

23

RNA and ATP substrates[6-11]. In vitro, DEAD-box proteins typically exhibit an RNA-dependent

24

ATPase activity that is associated with RNA duplex dissociation, helix formation, protein

25

displacement, or RNA secondary and tertiary structure rearrangements [6, 12, 13].

2

1 2

E. coli harbors five DEAD-box RNA helicases: DbpA, RhlB, SrmB, DeaD (CsdA), and RhlE [12,

3

14, 15]. Of these, SrmB has been implicated in the earliest stages of 50S ribosomal subunit

4

biogenesis. Deletion of SrmB confers a cold-sensitive phenotype and leads to an accumulation

5

of 40S particles corresponding to incompletely assembled 50S subunits[16]. SrmB forms a

6

specific ribonucleoprotein complex in vivo and in vitro with the r-proteins uL4 and uL24 and h11-

7

21 (nt 200-400) near the 5’ end of 23S RNA[17, 18] (Fig. 1d). Mutations in rRNA that suppress

8

the phenotype of ∆srmB were located far from the SrmB tethering site (Fig. 1e) but close to the

9

5S rRNA and 1024 G-ribowrench (which is a pseudoknot formed by helix 41 and an internal

10

loop between helix 41 and helix 42), as well as uL13 and the L7/12 stalk. The 1024 G-

11

ribowrench forms many contacts with uL13 and is a phylogenetically conserved pseudoknot[19].

12

One of the proposed functions of SrmB is to facilitate the folding of the 1024 G-ribowrench and

13

h42 structures in 23S rRNA without a requirement for ATP hydrolysis[18].

14 15

The helicase activity of SrmB on noncognate substrates in vitro requires a short stretch of

16

double stranded RNA with a long, single-stranded 5’ or 3’ overhang. It has been proposed that

17

long RNAs may be required to bridge individual SrmB monomers for dimerization[20], although

18

the oligomeric state of functional SrmB acting on ribosomal substrates is unknown. Recently, it

19

has been found that interactions with RraA, a protein that binds to RNase E and regulates its

20

endonucleolytic activity, can stimulate ATPase activity, although the roles of RraA and SrmB

21

together in vivo are unclear[21]. Another potential role for SrmB could be as an rRNA chaperone,

22

since SrmB is reported to have annealing activity[22]. More recent work by Iost and Jain [23]

23

identified ∆srmB suppressor mutations that map to the 5’-untranslated region of the uL13 and

24

uS9 operon that cause the overexpression of these two proteins and alleviate the cold-sensitive

25

phenotype of ∆srmB. These finding suggest a previously unknown function of SrmB in

3

1

regulating the expression levels of r-proteins and thus indirectly mediating ribosome assembly.

2

However, it is difficult to integrate the results from previous work into a mechanistic picture

3

including the precise nature of the ribosomal substrate for SrmB and the conformational

4

changes that it might catalyze through direct interactions with ribosome assembly intermediates.

5

Additionally, differences in strains, growth temperature, and growth medium previously reported

6

in the literature make it hard to pinpoint the SrmB function(s) in assembly (Table S1).

7 8

Previously, we explored the effects of limiting an essential protein, bL17, by quantitative mass

9

spectrometry (qMS) to determine r-protein occupancy and cryo-electron microscopy (cryo-EM)

10

to examine structures of intermediates[24]. We have also developed methods to quantitatively

11

measure the rRNA modification state [25]. Here, we us similar approaches to characterize the

12

40S intermediate present at 37°C in ∆srmB in the E. coli K-12 BW25113 strain grown in minimal

13

medium. Our decision to use 37°C is based on our de sire to facilitate comparison to the

14

structures of assembly intermediates resolved previously [24], and the choice of a defined

15

medium was driven by facilitation of isotope labeling (15N and 2H) for quantitative mass

16

spectrometry. Our results suggest a possible mechanism through which SrmB aids ribosome

17

assembly in vivo as an RNA chaperone to resolve misfolded and trapped RNA structures, and

18

by stabilizing early r-protein and rRNA binding events in ribosome assembly.

19 20

Results

21

Multiple ribosome assembly intermediate populations are present in ∆srmB

22

∆srmB has previously been shown to have a cold-growth defect in the E. coli K12 strain

23

WJW45[16], indicated by the presence of a 40S peak during sucrose gradient ultracentrifugation.

24

This defect is apparent in our E. coli ∆srmB BW25113 strain grown at 37°C (Fig. S1), as well,

25

although to a lesser extent; this defect at 37°C wa s also observed by Jagessar and Jain [26].

4

1

The composition of the 40S peak from ∆srmB BW25113 was characterized using a qMS protein

2

inventory experiment (Fig. S1, left workflow, and Fig. 2). The normalized protein levels for the

3

control WT 50S particle represent fully assembled 50S particles with stoichiometric ribosomal

4

protein occupancy (Fig. 2, black). On the other hand, three groups of r-proteins with differential

5

occupancy were observed in the ∆srmB 40S fraction (Fig. 2, pink). The first group, which

6

contains, uL1, uL3, uL4, uL5, bL17, uL18, bL20, bL21, uL23, uL24, and uL29 were all found at

7

levels that were stoichiometric compared to the control 50S particles. Many of these proteins

8

(uL1, uL3, uL4, bL20, bL21, uL23, and uL24) are primary binding proteins that bind directly to

9

the rRNA and nucleate ribosome assembly. The second group, with levels between 50 and 90%,

10

include proteins that are important for the interactions of the 30S and 50S (bL2, uL14, and bL19,

11

Fig. 1b.) The third group is composed of r-proteins that are present in less than 50% of the

12

population. These proteins include many of the late binding proteins[27] (uL6, uL7/12, bL9, uL10,

13

uL16, bL25, bL27, bL28, uL30, uL31, bL35, and bL36) as well as several early binding proteins

14

such as uL13 and intermediary binding protein bL32. Many of these proteins surround the

15

peptidyl transferase center (uL6, bL10, uL16, and uL31, Fig. 1c) and contain protein that are

16

important for the proper docking of the central protuberance (bL25, Fig. 1a). uL22 was not

17

identified in the 40S fraction. These data generally agree well with previous work by Charollais,

18

et al.[16], although there are differences between the two datasets that could be due to

19

differences in the strains, purification protocols, the temperature of the culture, or data collection

20

and analysis procedures.

21

whereas Charollais et al. use label-free quantification[16].

For example, our work here utilizes relative quantification[28],

22 23

Our qMS data suggest that there are multiple assembly intermediates in the ∆srmB 40S peak,

24

given the large number of proteins that were found in substoichiometric amounts (Fig. 2 group 2

25

and 3). Furthermore, the data suggest that the binding site for SrmB, formed by uL24 and uL4,

5

1

could be intact for all assembly intermediates in the 40S peak, as both are present at nearly

2

stoichiometric levels. Proteins uL5 and uL18 are present at WT levels, but protein uL25

3

necessary for proper integration of 5S rRNA and docking of the central protuberance is present

4

at substoichiometric levels. This could be explained by the presence of intermediates with a

5

misdocked or misfolded central protuberance. Based on the protein composition data, the

6

proposed SrmB binding site appears to be intact in the intermediates, but the 40S peak has a

7

large amount of compositional heterogeneity that is presumably also reflected in structural

8

heterogeneity.

9 10

Altered rRNA modification levels suggest effects of ∆srmB near the peptidyl transfer site.

11

E. coli 23S rRNA contains 25 modified nucleosides spread over the functionally important sites

12

of the ribosome, including the peptidyl-transferase center, exit tunnel, and the intersubunit

13

bridges.

14

hydroxycytidine, one dihydrouridine, and one methylated pseudouridine. Several of these

15

modifications were recognized as important elements contributing to translation and the 50S

16

assembly process[25, 29-31].

These include 10 base and 3 ribose methylations, 9 pseudouridines, one

17 18

We previously determined the relative order of 23S modifications in MRE600 E. coli cells using

19

a qMS analysis protocol[25]. By applying the same qMS workflow to the 40S samples from

20

∆srmB (Fig. S1), we identified and quantified 22 out of 25 known 23S modifications relative to

21

the 70S from WT BW25113 (Fig. 3). Based on these data there are three distinct groups of

22

modifications, introduced consecutively during 50S assembly in WT cells (only two groups were

23

resolved previously) (Fig. 3a and Fig. S2 for biological replicates). However, there are two

24

specific modification events in the ∆srmB dataset where the order of events is changed without

25

globally perturbing the whole pathway (Fig. 3b). When SrmB is present, Cm(2498) and

6

1

ho5C(2501) are introduced during intermediate stages of the 50S assembly process. In ∆srmB,

2

hydroxylation at 2501 by YdcPis largely delayed, and the ribose methylation at 2498 by RlmM

3

is accelerated with respect to other modifications in the wild type.

4 5

The decoupling between modifications at two closely spaced residues in the peptidyl

6

transferase center (at the base of h89, Fig 3c) of the 50S is surprising. We speculate that SrmB

7

is required for proper timing of Cm(2498) and ho5C(2501) modifications and possibly plays an

8

active role in displacing RlmM, which in turn allows 2501 to be hydroxylated. Furthermore,

9

close examination of our raw LC-MS data suggest presence of both 2501-modified and 2501-

10

unmodified RNase U2 fragments (Fig. S3). In the mature 70S, this residue is known to be

11

partially modified depending on the strain and growth conditions [32, 33]. The levels of

12

ho5C(2501) were estimated to be 85% in the wild type cells and 60-70% in ∆srmB ribosomes.

13

This reduction might be a direct consequence of the delay in the 2501 modification step when

14

SrmB is unavailable. In summary, our findings using ∆srmB suggest specific alterations in the

15

order of Cm(2498) and ho5C(2501) modifications and possibly in the assembly of the peptidyl

16

transferase center.

17 18

Cryo-EM of ∆srmB 40S reveals three assembly intermediates

19

Cryo-EM analysis of the particles in the 40S peak were performed using the same general

20

approach that was used for bL17 limitation strain as previously described [24]. Three distinct

21

classes were identified by 3D classification, at resolutions of 4.7-5.8Å (Fig S4.), from the

22

combined 40S fractions of the sucrose gradient ultracentrifugation experiments (Fig. 4). The

23

three classes are arranged in order of increasing maturity and represent 36% (A class, Fig. 4a),

24

39% (B class, Fig. 4b), and 25% (C class, Fig. 4c) of the classified particles, respectively. While

25

these resolutions are insufficient to confidently build models into the density, we did not use

7

1

additional processing and refinement strategies in order to increase resolution. The cryo-EM

2

density maps presented in Fig 4. have been filtered to 10Å in order to facilitate data analysis, as

3

we are interested in exploring broad structural features at the level of protein occupancies rather

4

than small conformational changes or subtle differences in individual amino acids or rRNA

5

bases. The low-pass filtered maps are accordingly used for subsequent analyses. The unfiltered

6

maps with the corresponding FSC curves are presented in Fig. S4 and were used to confirm

7

findings from the filtered dataset (see Table S2 for statistics). To broadly compare the maps, an

8

occupancy matrix was calculated based on quantitative comparison of the experimental map to

9

a reference map (PDB ID 4YBB), which has been segmented into a standard set of rRNA

10

helices and r-protein densities, as previously described[24]. By clustering the protein and rRNA

11

helix occupancy, we observed five major structural blocks that assemble cooperatively (Fig. 4d).

12 13

For the most part, the protein abundances observed in the qMS data (Fig. 2, pink) match well to

14

the protein occupancies observed in the cryo-EM dataset (Fig. 4d). Differences between the

15

qMS data and the cryo-EM occupancy analysis can be caused by several reasons. First, r-

16

proteins that are present in the qMS data may not appear in the cryo-EM density because they

17

are bound to flexible structural elements that are not resolved by cryo-EM. Conversely, proteins

18

present in the cryo-EM maps that have low abundance by qMS can be explained by non-native

19

structures, such as misfolded rRNAs, misdocked r-protein binding, or unidentified cellular

20

factors bound., In fact, the reference-based nature of the cryo-EM occupancy analysis is blind

21

to non-native features. Furthermore, qMS is a bulk measurement of the average of protein

22

occupancy of many different intermediates, while the cryo-EM occupancies are calculated for

23

the individual solved intermediate structures. Therefore, proteins that are substoichiometric by

24

the qMS analysis can be found in one or more of the observed cryo-EM intermediates. Finally,

25

some proteins, like bL31, uL1, and uL7/12, are not included in the 4YBB crystal structure or, like

26

bL9, are in different conformations due to the crystallization process. Regardless, the cryo-EM

8

1

occupancy analysis provides a quantitative means by which to interpret structural similarities

2

and differences of the ribosome assembly states For example, by mapping the blocks revealed

3

by the occupancy analysis to the 2D- structure of the 23S (Fig. 4e), we observe that the folding

4

blocks map to multiple rRNA domains, that are connected by tertiary contacts. We have

5

previously observed this “block” behavior in our work under the r-protein bL17 limitation[24].

6 7

The first block of assembly (Fig. 4e and Fig. S5) corresponds to proteins and rRNA helices that

8

have >90% density observed in all three cryo-EM maps, which includes the SrmB binding site

9

(Fig. 5, row 1). This block also supports a fully-formed peptide exit tunnel and contains most of

10

the rRNA helices from Domains I and III. Block 2 (Fig. 4d,e and Fig. S5, yellow) represents

11

proteins and rRNA helices that are present in the B and C classes, but are not fully occupied in

12

the A class. bL20, bL21, uL15 and many of the rRNA helices in Domain II belong to this class,

13

thus indicating that some of the earliest r-proteins and helices necessary for stabilizing the 5S

14

rRNA are not stable in the A class. In block 3 (Fig. 4d,e and Fig. S5, green) are proteins and

15

helices that are almost fully occupied in the A and C classes, but not fully occupied in the B

16

class, which include rRNA elements of Domain V, bL28, and bL35. Closer inspection of the A

17

class reveals that much of this density is due to misdocked rRNA or r-protein structures that do

18

not conform to the density that would be expected for the native elements (according to PDB ID

19

4YBB), confirming that the A class is less mature than the B class.

20 21

Block 3 also contains occupancy at the position of uL13, which is present at a low stoichiometry

22

in the qMS data. Upon closer examination of this region in the maps, we observe that there is a

23

“bridge” of non-native rRNA density spanning from the base of the uL7/12 stalk to the uL13

24

binding site in the B class and the C class (Fig. S6) and non-protein density in the A class (Fig.

25

5, row 2).

26

seemingly anomalous density observed in the cryo-EM occupancy analysis. Block 4 (Fig. 4d,e

This bridge partially occludes the uL13 binding site, thus accounting for the

9

1

and Fig. S5, blue) represents proteins and rRNA helices that do not have any occupancies in

2

the cryo-EM maps. These proteins and rRNA helices involve late formation of the peptidyl

3

transfer center (h89, h91) and important sites of interactions between the 30S and 50S

4

subunits, such as (h68, h69, h71) uL16 and h42. Others are important for the formation of the

5

uL7/12 stalk (uL10, uL11, h42-44),. Block 5 (Fig. 4d,e and Fig. S5, purple) represents the

6

central protuberance proteins (L5,L18, L25) 5S rRNA, and helixes 38, 83-85 which directly

7

contact 5S RNA, which is only stable enough for density to be observed in the C class. Although

8

we only observe density for the central protuberance in the C class, qMS data suggests that the

9

central protuberance is at least partially formed, but flexible and misdocked, as many of the r-

10

proteins necessary for stable central protuberance docking are observed in the qMS data but

11

the density does not average into a structure in the cryo-EM data.

12 13

This analysis, combined with qMS data, reveals that the 40S peak is conformationally and

14

compositionally heterogenous. The three structurally distinct can be naturally organized into a

15

putative linear assembly pathway of A->B->C->50S, proceeding from the least to the most

16

mature intermediate, in the absence of SrmB. The SrmB-B class and SrmB-C class are broadly

17

similar to the major L17-C classes and L17-E classes from the bL17-limitation strain data,

18

implying that these structures are likely common on-pathway intermediates for further

19

maturation, whereas the presence of the SrmB-A class is unique to SrmB deletion. Although the

20

peptidyl exit site seems to be well-formed for all three classes, there are severe defects in the

21

peptidyl transferase center, the central protuberance, and intersubunit bridge contacts.

22

Interestingly, many of structural perturbations center along the sites of misdocked or missing

23

density for rRNAs that suppress SrmB deletion (Fig. 5, row 3). Furthermore, there is also a

24

“bridge” of non-native density between h97 and h41 that could be due to non-native RNA

25

tertiary interactions or could be an extension of local rRNA helices (Fig. 5, row 4).

26 10

1

Discussion

2

The SrmB binding site is formed in the absence of SrmB

3

The SrmB binding site, which is composed of uL4, uL24, and nt 200-400 (h11-21) [17] is intact

4

in all three cryo-EM structures (Fig 5. row 1) and uL4 and uL24 are at stoichiometric levels in

5

the protein qMS data, which indicates that the SrmB binding site has no clear deficiencies

6

during the assembly process at 37°C. Thus, the earl iest nucleation stages of ribosome

7

assembly are able to proceed without the intervention of SrmB. Our observations are fully

8

consistent with previous studies by the Dreyfus group [14, 16-18, 20] and suggest that SrmB

9

acts distally to its binding site by acting as a chaperone for proper rRNA docking (e.g. h42) and

10

r-protein binding events (e.g. uL13 and uL25, Fig 5. row 2, Fig. 6b) or by providing rigidity to the

11

nascent assembling ribosome so that later steps of assembly can occur on a solid base (Fig.

12

6c).

13 14

Structural defects near ∆srmB suppressor sites, at peptidyl transfer center and the uL7/12 stalk

15

are present in ∆srmB

16

Density is missing for uL13 and helix 42 in all three of our cryo-EM maps, and bL25 is only

17

present in the C class (Fig. 5, row 2). These structural elements are located near the proposed

18

site of action for SrmB (Fig. 5, row 3). uL13 directly interacts with the 1024 G-ribowrench and

19

bL25 interacts with h42. Given that uL13 and bL25 are at levels three times lower than their

20

corresponding levels in WT 50S ribosomes in the qMS data, it likely that these proteins are not

21

present in the ribosome assembly intermediates, and we observe non-native densities that

22

would not allow for proper protein docking. These non-native densities form a “bridge” that could

23

be an extension or alternative conformation of h41 or h97 (Fig. 5 row 4). h41, which participates

24

in 1024 G-ribowrench formation, is slightly misdocked, although density is clearly observed for

25

bL20 and bL21, both of which contact the outside of this helix. h97, while maintaining at least

11

1

50% occupancy in all three classes, is not well-resolved at the area where the “bridging” density

2

occurs. Furthermore, the r-proteins and rRNAs that support the uL7/12 stalk are missing in all

3

three structures (L11, L10, h42- h44, Fig 4d-e ), indicating that SrmB plays a key role in its

4

formation.

5 6

The changes in the order of Cm(2498) and ho5C(2501) modifications suggest alternative

7

conformations of the rRNA in the peptidyl transferase center region, which alter the availability

8

of rRNA for modification. These modifications occur at the base of h89, which is not resolved in

9

any of the cryo-EM data, although the C class exhibits extra density at the base of h89. These

10

data suggest that there are severe defects at the peptidyl transferase center. Defects in peptidyl

11

transferase center formation were previously observed in 50S assembly intermediates isolated

12

with other strains, including bL17-lim [24, 34, 35] and are thought to

13

intermediate particles, if mistakenly integrated into a 70S ribosome, are unable to translate

14

proteins.

ensure that these

15 16

SrmB alleviates kinetic traps for ribosome assembly

17

Our data qMS and cryo-EM data reveal that, in the absence of SrmB, there are multiple sites of

18

defects in ribosome assembly: the peptidyl transferase center (Fig. 1b), the central

19

protuberance (Fig. 1a), and areas of contact between the 30S and 50S subunits (Fig. 1c), and

20

L7/L12 stalk. Although SrmB deletion is less severe for ribosome assembly at 37°C, compared

21

to cold temperatures (<30°C), ribosome assembly pro ceeds with the accumulation of

22

intermediates (classes A-C). Overall, our data suggest that it is likely that one of the functions of

23

SrmB is to act against rRNA falling into a misfolded kinetic trap (Figure 6).

24 25

Given the data presented here, along with the current literature, it is likely that SrmB has

26

multiple roles depending on the stress conditions of the cell. First, given the recent work by Iost

12

1

and Jain [23], SrmB acts in a mechanism to regulate uL13 production, and the 40S particles

2

thus represent a “uL13-limited” assembly pathway(Fig. 6b). However, our whole cell proteomics

3

analysis (Fig. S7) revealed that there is no significant depletion (~10% reduction with respect

4

to other r-proteins in ∆srmB ) of uL13 at either 37°C or 18°C, and so it i s unlikely that a “uL13-

5

limited” assembly pathway is represented in our data. Second, given that in vitro, SrmB can

6

facilitate structural rearrangements without ATP hydrolysis[18, 22], we hypothesize that the

7

chaperone function of SrmB are through its C-terminal tail (Fig. 6a) which facilitates correct

8

docking ofthe central protuberance and the peptidyl transferase center . C-terminal tail may also

9

assist , uL13, bL25, and h42 , and they would be able to form their proper contacts and dock

10

the rest of the rRNA and r-proteins into a fully-functional 50S (Fig. 6c). However, given that the

11

C-terminal tail may not be necessary for ribosome assembly at 30°C[17] (at least in the context

12

of SrmB overexpressed from a plasmid), it is possible that SrmB has a different mechanism of

13

action under cold-stress conditions.

14

interactions of uL24, uL4, and nt200-400. The rigidity provided by these initial interactions could

15

prevent non-productive rRNA structures from forming, thus ensuring the success of ribosome

16

assembly, even at colder temperatures, where rRNA could become trapped in unfavorable

17

intermediary states (Fig. 6d). Third, SrmB could act as a nexus for other proteins, such as RlmM

18

and the newly discovered YdcP responsible for ho5C2501 [36] to bind in the correct order, thus

19

ensuring that ribosome rRNA modifications occur at the proper times (Fig. 6e). The evidence

20

from our qMS, rRNA modification MS, and cryo-EM data from ∆srmB E. coli grown at

21

physiological temperatures lead to the conclusion that SrmB anchors to the uL24, uL4, and nt

22

200-400 region in order to perform chaperone activities through physical interactions with

23

ribosome assembly intermediates. However, it seems likely that SrmB plays multiple roles in

24

ribosome assembly depending on the stress conditions of the cell.

Alternatively, SrmB could act to stabilize the nascent

25

13

1

Acknowledgements

2

The authors would like to thank Dr. V. Patsalo for help with whole cell proteomics analysis and

3

Dr. J Hammond for helpful discussions and review of this paper. This work was supported by a

4

grant from the NIH R01-GM053757 to JRW, the NIH DP5-OD021396 to DL, the NIH F32-

5

GM111013 to AMP, the NIH F32-GM103173 to CFS, and NSF DGE-1346837 to EMG.

6 7

Materials and Methods

8

Bacterial Strains and plasmids

9

WT BW25113 E. coli and the ∆SrmB BW25113 E. coli strain from the Keio Knockout Collection,

10

were obtained from the E. coli Genetic Stock Center [37].

11 12

Cell growth

13

∆srmB cells were grown at 37°C in M9 glucose minimal med ium supplemented with trace

14

metals in the presence of either 1 g/L of

15

whole cell proteomics analysis) or using a mixture of 0.5 g/L 14N ammonium sulfate and 0.5 g/L

16

15

17

facilitate isotope labeling and relative quantitation of ribosomal proteins and RNA modifications’

18

∆srmB BW25113 cultures were grown to OD600 0.5–0.6, were quenched on an equal volume of

19

ice and were harvested by centrifugation at 5000 rpm for 15 min. Cell pellets were either

20

immediately lysed or stored at −80 °C prior to lysis.

14

N ammonium sulfate (EM, RNA modification and

N ammonium sulfate ( QMS protein inventory). This was the sole nitrogen sources in order to

21 22

Sucrose Gradient Purification of Ribosomal Particles

23

Frozen cell pellets for ∆srmB cultures were then thawed and resuspended in 20 mM Tris–HCl,

24

pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM

25

dithiothreitol [DTT], 1 mM phenylmethylsulfonylfluoride (PMSF), and 20 U/ml DNase I (Sigma),

14

1

and EDTA-free protease inhibitor cocktail (Roche Applied Science). Cells were lysed in a bead

2

beater (BioSpec Products, Inc., Bartlesville, OK) using 0.1-mm zirconia/silica beads (3x40

3

second pulses with 2 minutes on ice in between). Insoluble debris was removed by two

4

centrifugation steps: a low-speed spin at 6000 rpm for 10 min and then a high-speed spin

5

centrifugation step at 16,000 rpm (31,000g) for 40 min.

6 7

The clarified supernatant was loaded onto a 13–51% (w/v) or 10-40% (w/v) non-dissociating

8

linear sucrose gradient (50 mM Tris–HCl, pH 7.8, 10 mM MgCl2, and 100 mM NH4Cl) and

9

centrifuged in a Beckman SW32 rotor at 26000 rpm for 18 or 16 hours at 4°C. For protein

10

inventory QMSanalysis, approximately 84 fractions were collected from each sucrose gradient

11

using a Brandel gradient fractionator. Based on the UV 254 nm trace, gradient fractions

12

corresponding to the 40S ribosome peak were pooled together. For EM analysis, the combined

13

fraction was diluted to 3X volume in gradient buffer (50 mM Tris–HCl, pH 7.8, 10 mM MgCl2,

14

and 100 mM NH4Cl) and buffer exchange 3 times using a 3 kDa cutoff concentrator (Amicon) to

15

remove most of the sucrose The concentrated to 50 µl sample was used to estimate RNA

16

concentration using OD260.

17 18

ESI-TOF Mass Spectrometry

19

Each experiment was performed in triplicate. The data show a representative replicate (Fig. 2).

20

For protein inventory experiments, 50 pmol of both

21

50 pmol of 40S subunit purified via sucrose gradient centrifugation. 70S from WT cells were

22

purified using a sucrose cushion.

23

trichloroacetic acid (TCA). The protein precipitate was pelleted by centrifugation at 13,000g for

24

30 min at 4 °C. The supernatant was removed, and th e pellets were rinsed first with 10% TCA

25

and then with ice- cold acetone, dried in a Speed-Vac concentrator, and then resuspended in 40

26

µL of 100 mM ammonium bicarbonate (pH 8.5) in 5% acetonitrile (ACN). A 4-µL aliquot of 50

15

N WT-70S and 14N WT-70S were added to

Samples were incubated on ice for 12 h in 13%

15

1

mM DTT was added, and the samples were incubated at 65 °C for 10 min. Cysteine residues

2

were modified by the addition of 4 µL of 100 mM iodoacetamide followed by incubation at 30 °C

3

for 30 min in the dark. Proteolytic digestion of the proteins was carried out by the addition of 4

4

µL of 0.1 µg/mL sequencing grade porcine trypsin (Promega, Co., Madison, WI) with incubation

5

overnight at 37 °C. Undigested proteins were precip itated by adding 1/3 volume of 20% ACNin

6

2% trifluoroacetic acid and removed by centrifugation. The supernatant was loaded to a

7

PepClean C18 spin column (Thermo Fisher Scientific Inc., Rockford, IL) to remove salts and

8

concentrate the samples. The eluant was dried in a Speed-Vac concentrator and the peptides

9

were re-dissolved in 10 µL of 5% ACN in 0.1% formic acid. An 8 µL of aliquot was used for the

10

electrospray ionization time of flight (ESI-TOF) analysis.

11 12

The peptide samples were analyzed on an Agilent 1100 Series HPLC instrument coupled to an

13

Agilent ESI-TOF instrument with capillary flow electrospray (Agilent Technologies Inc., Santa

14

Clara, CA). The digested ribosomal proteins were injected using an autosampler onto an Agilent

15

Zorbax SB C18 150mm × 0.5mm HPLC column. The mobile phases used were buffer A (H2O,

16

0.1% formic acid) and buffer B (acetonitrile, 0.1% formic acid). Peptides were separated at a

17

flow rate of 7 µL/min using the linear gradient( step 1: 5–15% buffer B over 10 min; step 2: 15–

18

50% buffer B over 70 min, and step 3: 50–95% buffer B over 4 min). Data were collected using

19

positive polarity over the m/z range of 300–1300.

20

Quantitative mass spectrometry data was processed and analyzed as previously described[38-

21

41]

22 23

Whole cell Proteomics

24

Whole cell relative abundances of the r-proteins were obtained by culturing ∆srmB and WT

25

cells in M9 at 37°C or at 18°C.

26

WT cells respectively. ∆srmB and WT cells were mixed at ~ 1:1 ratio based on the OD

14

N- and

15

N-ammonium sulfate were used to label ∆srmB and

16

1

measurements, lysed overnight in 20% TCA at 4°C, an d the precipitated proteins digested with

2

trypsin as described above. LC-MS/MS data were collected on a Sciex 5600+ Triple TOF

3

coupled to the Eksigent expert nanoLC [24].

4

normalized to the medium over ~2500 E. coli peptides identified and quantified in each sample

5

(Fig. S7).

14

N/15N ratios for each ribosomal peptide were

6 7 8

Analysis of 23S RNA modifications.

9

MS-based quantitative measurements of 23S rRNA modifications were done as described in the Wild-type and ∆srmB rRNAs were metabolically labeled with

14

15

10

prior work [25].

11

ammonium sulfate in the presence of 5,6-D-uracil (Cambridge Isotope Laboratories) enabling

12

identification of pseudouridines in both the sample and the reference. Abundances of individual

13

modifications were assessed using nucleolytic fragments detected using RNase T1, A, or U2

14

cleavages, relative to their abundances in the 23S standard purified from the mature WT

15

ribosomes:

N- or

N-

16 Relative modification level =

Peak Intensity of ∆ Peak Intensity of ∆ + Peak Intensity of WT

17

where the ∆srmB and WT are differentially labeled with 14N and 15N isotopes.

18

Two additional U2/T1 digestion fragments were added to the previously reported list of 23S

19

specific oligonucleotides[25]: 2604-ΨΨCG-2607, used to monitor Ψ(2604) and Ψ( 2605), and

20

chemically identical 2456-CΨG-2458 and 2579-CΨG-2581, reporting on the presence of both

21

Ψ(2457) and Ψ( 2580).

22 23

Electron Microscopy Sample Preparation and Data Acquisition

17

1

Gold grids were a generous gift from Dr. Bridget Carragher and were prepared as in [42].

2

Sample was diluted to ~225 nM in gradient buffer and applied to a plasma cleaned (6s, Gatan

3

Solarus) gold grid in humidified CP3 chamber (FEI). Sample was blotted automatically for 2.5s

4

and plunged into liquid ethane, then stored in liquid nitrogen until imaged.

5 6

Data were collected on an Thermo Fisher Scientific (formerly FEI) Titan Krios electron

7

microscope operating at 300 kV equipped with a Gatan K2 Summit detector using the Leginon

8

software [43] with an estimated underfocus ranging from 1.0 µm to 3.5 µm (distributed in an

9

approximately Gaussian manner).

The total dose was 45 e-/ Å2, fractionated over 50 raw

10

frames collected over a 10 second exposure time (200 ms per frame), with each frame receiving

11

a dose of ~7 e-/ Å2. 1958 movies were recorded at a calibrated magnification for the position of

12

the detector of 38,167 (nominal magnification of 22,500), corresponding to a pixel size 1.31 Å.

13

To overcome problems of preferred orientation on the grid, particles were imaged at different tilt

14

angles (0, 10, 20, 30, 40, 50 degrees) to obtain different views [44].

15 16

Image Processing

17

All pre-processing was performed within the Appion pipeline[45] and individual programs used

18

within the pipeline are cited below. Frames were aligned using MotionCor2[46], and then used

19

for processing. All micrographs were manually masked using the masking tool to remove

20

regions corresponding to the gold grid bars and large aggregates.

21

function (CTF) for all micrographs was estimated using CTFFind3 and CTFTilt[47]. 284,420

22

particles were selected using low-pass filtered 50S ribosomal subunit templates from the

23

micrographs using the FindEM package [48]. A phase-flipped, contrast-inverted, 2x-binned

24

stack was created from these picks with a box size of 128 and pixel size 2.62 Å and was used

25

until the final 3D refinements. Reference-free 2D alignment of this stack was accomplished

26

using ML2D [49] followed by RELION [50]. The 2D classes were visually inspected, and any

The contrast transfer

18

1

classes that were clearly 30S, 70S, or other known cellular structures were removed. The

2

remaining 199,381 particles were sorted using projection-matching into one of seven 3D maps

3

(30S, 70S, and five different 50S assembly intermediates obtained during pre-processing). The

4

129,271 particles that were matched to the 50S assembly intermediates were then subjected to

5

3D classification in RELION using 5 classes [50] and a 50S map filtered to 60 Å as an initial

6

model. After removing classes that either did not produce an interpretable map or clearly

7

belonged to a 30S or 70S particle (and was obscured in the prior, coarse-grained classification),

8

60,488 particles remained and resulted in three broadly different maps comprising three classes

9

described herein (Fig. 4). The angles and class occupancies were refined within Frealign[51].

10

Final maps were sharpened using cisTEM [52] by flattening the amplitude spectrum between

11

10Å and the resolution of the individual maps.

12 13

Occupancy Matrix Analysis

14

The relative occupancy of

15

calculated using a combination of 3rd party programs and in-house scripts. The experimental

16

maps were prepared for quantitative comparison to a reference map generated from PDB entry

17

4YBB. Briefly, the unsharpened, unmasked maps from Frealign were filtered to 10Å, and the

18

standard deviation (σ) of voxel values for each map was calculated using relion_image_handler,

19

as an estimate of the noise in the maps. The average σ for the three final maps was 2.25±0.22.

20

The real-valued voxels for the maps were binarized to 0 or 1 using the relion_image_handler

21

command, with the threshold for binarization set to 3*σ for each individual map without any soft

22

edges. The reference map from the E. coli 50S subunit crystal structure (PDB ID 4YBB) was

23

segmented into 140** elements comprised of individual ribosomal proteins and of rRNA helices

24

according to 23S secondary structure [53]. Theoretical density of 4YBB was then calculated for

25

each element at 10 Å using the pdb2mrc command from EMAN[54]. The density for each voxel

ribosomal proteins and rRNA helices

n ∆srmB classes was

19

1

was then binarized as 0 or 1 using a threshold value of 0.016, which is the threshold that gave

2

approximately correct molecular weight values for individual r-proteins and rRNA helices. Finally,

3

the relative volumes in the binarized experimental and reference maps were calculated for each

4

of the 140 reference elements, resulting in a fractional occupancy between 0 and 1 for each

5

element. Observed occupancy values were then clustered across rows (classes) and columns

6

(rRNA/protein elements) using unsupervised hierarchical clustering, with a squared Euclidean

7

distance metric and Ward’s linkage method, implemented in Mathematica[57].

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

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[13] Linder P, Jankowsky E. From unwinding to clamping—the DEAD box RNA helicase family. Nature reviews Molecular cell biology. 2011;12:505. [14] Iost I, Dreyfus M. DEAD-box RNA helicases in Escherichia coli. Nucleic acids research. 2006;34:4189-97. [15] Jarmoskaite I, Russell R. RNA helicase proteins as chaperones and remodelers. Annual review of biochemistry. 2014;83:697-725. [16] Charollais J, Pflieger D, Vinh J, Dreyfus M, Iost I. The DEAD-box RNA helicase SrmB is involved in the assembly of 50S ribosomal subunits in Escherichia coli. Molecular microbiology. 2003;48:1253-65. [17] Trubetskoy D, Proux F, Allemand F, Dreyfus M, Iost I. SrmB, a DEAD-box helicase involved in Escherichia coli ribosome assembly, is specifically targeted to 23S rRNA in vivo. Nucleic acids research. 2009;37:6540-9. [18] Proux F, Dreyfus M, Iost I. Identification of the sites of action of SrmB, a DEAD-box RNA helicase involved in Escherichia coli ribosome assembly. Molecular microbiology. 2011;82:30011. [19] Steinberg SV, Boutorine YI. G-ribo motif favors the formation of pseudoknots in ribosomal RNA. Rna. 2007;13:1036-42. [20] Bizebard T, Ferlenghi I, Iost I, Dreyfus M. Studies on three E. coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases. Biochemistry. 2004;43:7857-66. [21] Pietras Z, Hardwick SW, Swiezewski S, Luisi BF. Potential regulatory interactions of Escherichia coli RraA protein with DEAD-box helicases. Journal of Biological Chemistry. 2013:jbc. M113. 502146. [22] Zhao X, Jain C. DEAD-box proteins from Escherichia coli exhibit multiple ATP-independent activities. Journal of bacteriology. 2011. [23] Iost I, Jain C. A DEAD-box protein regulates ribosome assembly through control of ribosomal protein synthesis. Nucleic Acids Res. 2019. [24] Davis JH, Tan YZ, Carragher B, Potter CS, Lyumkis D, Williamson JR. Modular assembly of the bacterial large ribosomal subunit. Cell. 2016;167:1610-22. e15. [25] Popova AM, Williamson JR. Quantitative analysis of rRNA modifications using stable isotope labeling and mass spectrometry. Journal of the American Chemical Society. 2014;136:2058-69. [26] Jagessar KL, Jain C. Functional and molecular analysis of Escherichia coli strains lacking multiple DEAD-box helicases. Rna. 2010;16:1386-92. [27] Chen SS, Williamson JR. Characterization of the ribosome biogenesis landscape in E. coli using quantitative mass spectrometry. Journal of molecular biology. 2013;425:767-79. [28] Sperling E, Bunner AE, Sykes MT, Williamson JR. Quantitative analysis of isotope distributions in proteomic mass spectrometry using least-squares Fourier transform convolution. Analytical chemistry. 2008;80:4906-17. [29] Sloan KE, Warda AS, Sharma S, Entian K-D, Lafontaine DL, Bohnsack MT. Tuning the ribosome: The influence of rRNA modification on eukaryotic ribosome biogenesis and function. RNA biology. 2017;14:1138-52.

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[30] Arai T, Ishiguro K, Kimura S, Sakaguchi Y, Suzuki T, Suzuki T. Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly. Proceedings of the National Academy of Sciences. 2015;112:E4707-E16. [31] Kipper K, Sild S, Hetényi C, Remme J, Liiv A. Pseudouridylation of 23S rRNA helix 69 promotes peptide release by release factor RF2 but not by release factor RF1. Biochimie. 2011;93:834-44. [32] Andersen TE, Porse BT, Kirpekar F. A novel partial modification at C2501 in Escherichia coli 23S ribosomal RNA. Rna. 2004;10:907-13. [33] Purta E, O'connor M, Bujnicki JM, Douthwaite S. YgdE is the 2′-O-ribose methyltransferase RlmM specific for nucleotide C2498 in bacterial 23S rRNA. Molecular microbiology. 2009;72:1147-58. [34] Bokov K, Steinberg SV. A hierarchical model for evolution of 23S ribosomal RNA. Nature. 2009;457:977. [35] Jomaa A, Jain N, Davis JH, Williamson JR, Britton RA, Ortega J. Functional domains of the 50S subunit mature late in the assembly process. Nucleic acids research. 2013;42:3419-35. [36] Kimura S, Sakai Y, Ishiguro K, Suzuki T. Biogenesis and iron-dependency of ribosomal RNA hydroxylation. Nucleic acids research. 2017;45:12974-86. [37] Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006;2:2006 0008. [38] Sykes MT, Shajani Z, Sperling E, Beck AH, Williamson JR. Quantitative proteomic analysis of ribosome assembly and turnover in vivo. Journal of molecular biology. 2010;403:331-45. [39] Sykes MT, Sperling E, Chen SS, Williamson JR. Quantitation of the ribosomal protein autoregulatory network using mass spectrometry. Analytical chemistry. 2010;82:5038-45. [40] Chen SS, Williamson JR. Characterization of the ribosome biogenesis landscape in E. coli using quantitative mass spectrometry. Journal of molecular biology. 2013;425:767-79. [41] Chen SS, Sperling E, Silverman JM, Davis JH, Williamson JR. Measuring the dynamics of E. coli ribosome biogenesis using pulse-labeling and quantitative mass spectrometry. Molecular bioSystems. 2012;8:3325-34. [42] Russo CJ, Passmore LA. Ultrastable gold substrates: properties of a support for highresolution electron cryomicroscopy of biological specimens. Journal of structural biology. 2016;193:33-44. [43] Suloway C, Pulokas J, Fellmann D, Cheng A, Guerra F, Quispe J, et al. Automated molecular microscopy: the new Leginon system. J Struct Biol. 2005;151:41-60. [44] Tan YZ, Baldwin PR, Davis JH, Williamson JR, Potter CS, Carragher B, et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat Methods. 2017;14:793-+. [45] Lander GC, Stagg SM, Voss NR, Cheng A, Fellmann D, Pulokas J, et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. Journal of structural biology. 2009;166:95-102. [46] Zheng SQ, Palovcak E, Armache J-P, Verba KA, Cheng Y, Agard DA. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods. 2017;14:331.

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[47] Mindell JA, Grigorieff N. Accurate determination of local defocus and specimen tilt in electron microscopy. Journal of structural biology. 2003;142:334-47. [48] Roseman AM. FindEM--a fast, efficient program for automatic selection of particles from electron micrographs. J Struct Biol. 2004;145:91-9. [49] Scheres SH, Valle M, Nunez R, Sorzano CO, Marabini R, Herman GT, et al. Maximumlikelihood multi-reference refinement for electron microscopy images. J Mol Biol. 2005;348:139-49. [50] Scheres SH. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol. 2012;180:519-30. [51] Grigorieff N. FREALIGN: high-resolution refinement of single particle structures. Journal of structural biology. 2007;157:117-25. [52] Grant T, Rohou A, Grigorieff N. cisTEM, user-friendly software for single-particle image processing. Elife. 2018;7. [53] Petrov AS, Bernier CR, Gulen B, Waterbury CC, Hershkovits E, Hsiao C, et al. Secondary structures of rRNAs from all three domains of life. PLoS One. 2014;9:e88222. [54] Ludtke SJ, Baldwin PR, Chiu W. EMAN: semiautomated software for high-resolution singleparticle reconstructions. J Struct Biol. 1999;128:82-97. [57] Wolfram Research, Inc., Mathematica, Version 12.0, Champaign, IL (2019).

19 20 21 22 23 24 25 26 27

Figure Legends

28 29

Fig. 1. Anatomy of the 50S subunit (PDB ID 4YBB) including (a) the uL1 stalk (orange), CP

30

(red), and L7/12 stalk (yellow); (b) areas of contact between the 50S and 30S subunits (blue);

31

(c) helices involved in the peptidyl tranferase center (red, individual amino acids involved

32

colored dark red); (d) the SrmB binding site (rRNA in dark green, proteins uL4 and uL24 in

23

1

green and light green, respectively); and (e) mutations that can ameliorate ∆srmB (orange)

2

which occur near the region of uL13 (red) and uL25 (dark red) binding.

3 4

Fig. 2. Relative quantification of 40S from ∆srmB (pink) and WT 50S ribosome particles (black).

5

The relative 50S uL20 protein levels were normalized to 1, and remain consistent throughout

6

the experiment. (Pink) Relative levels of proteins in the 40S intermediate. The proteins fall into

7

three groups: less than 0.4 (gray), between 0.4 and 0.92 (medium gray), and greater than 0.92

8

(dark gray). If no peptides were found, the protein is represented by an x; otherwise, data was

9

taken in triplicate, data from only one peptide are shown as an open circle, and data from more

10

than one peptide are shown as closed circles.

11 12

Fig. 3. Quantitative MS reveals the relative order of individual 23S rRNA modification events. (a)

13

Sucrose density gradient profile of WT. Fractions are denoted by vertical lines. Relative rRNA

14

modification levels were calculated across the sucrose gradient and normalized to the levels of

15

unmodified 23S in each fraction. Modifications are classified as early (red), intermediate (green),

16

or late (cyan). (b) Sucrose density gradient profile of ∆srmB. Cm(2498) and ho5C(2501), which

17

differ in order between WT and ∆srmB strains, are labeled for each plot shown. (c) Relative

18

position of C2498 and C2501 within the crystal structure of the bacterial ribosome. r-proteins are

19

shown in grey. Helix 89 of the 23S rRNA, which contains C2498 (red spheres) and C2501(cyan

20

spheres), is shown in yellow. Helices 41-44 of the 23S rRNA, which contain the sites of

21

mutations that stabilize the growth defect at cold temperatures in ∆srmB strain (A1039 and

22

A1027) are in green. C27 is the site of another stabilizing mutation in the 5S rRNA (grey).

23

Structure used was PDB ID 4YBB.

24 25

Fig. 4. Analysis of the three 40S intermediate cryoEM structure. All cryoEM classes have been

26

filtered to 10Å and are shown with a soft mask. (a) The least mature class: A class. (b) The

24

1

intermediate class, B class. (c) The most mature class observed in the data, the C class. Blocks

2

of structural elements in (a-c) are colored according to the occupancy analysis in d. (d)

3

Occupancy analysis of the r-proteins and rRNA helices as referenced to PDB ID 4YBB. The

4

largest Block 1 (Fig. S5) is not shown here, and has 100% occupancy across all structures. (e)

5

Structural elements of the rRNA helices defined by the occupancy analysis mapped on to the

6

2D 23S rRNA domain structure. Colors correspond to the blocks in (d). Block 1 is shown in red.

7

rRNA domain contacts that span across domains are shown in solid lines.

8 9

Fig. 5. Specific areas pertinent to SrmB binding in reference to the theoretical cryoEM density of

10

4YBB. From left to right: the A class, B Class, C class, and the theoretical 4YBB EM

11

representations, and the 4YBB model (right column) are represented in gray. The original, non-

12

filtered cryoEM densities are used for visualization. Color scheme for elements in each row is at

13

the end of the row and in the first column. (Row 1) The SrmB binding site. L4 is colored in olive

14

green, L24 is colored in lime green, and nt200-400 are colored in sea green. (Row 2) Sites of

15

known SrmB deletion suppressor mutations. h42 is colored in light blue, h41 is in cyan and the

16

region of the 5S is colored in light steel blue. (Row 3) Areas of known disruption in ∆srmB. uL13

17

colored in red, bL25 in dark red, h42 is dark orange, and the dark red arrow points to density

18

blocking uL13 binding. (Row 4) Areas of non-native density that could be extensions of local

19

rRNA helices. h41 is in purple, bL20 is in magenta, bL21 is in dark magenta, and h97 is in pink.

20 21

Fig. 6. Potential pathways for SrmB to act in ribosome assembly. (a) Sequence of the C-

22

terminal tail of SrmB. The blue box indicates predicted α-helix content, otherwise, the rest of the

23

C-terminus is predicted to be uncoiled. Red stars indicate positively charged amino acids that

24

could participate in RNA binding. The predicted minimum length of an α-helix, followed by

25

random coils, has a minimum end-to-end length of 112Å, which is long enough to span the front

26

of the ribosome from the uL1 stalk to the uL7/12 stalk. (b) SrmB could regulate the expression

25

1

of uL13, which causes the ribosome assembly pathway to stall under cold-stress conditions (c)

2

SrmB could stabilize the uL4:uL24:nt 200-400 interactions, thus providing a stable base that

3

could prevent misfolding in further rRNA folding events. In this case, it is only the binding of

4

SrmB that stabilizes the rRNA, and the C-terminal tail is not needed for further structural

5

rearrangements. (d) The C-terminal tail of SrmB could act as a folding chaperone to ensure

6

proper central protuberance docking and rRNA folding around the uL13 binding site after SrmB

7

docks into its binding site. (e) SrmB could also act as a dock for other proteins, either by means

8

of its C-terminal tail or by some other protein: protein interactions. In this case, SrmB would

9

guide the rRNA into the proper conformations for docking and release of other proteins, such as

10

methyltransferases (such as RlmM) or hydroxylases.

11 12

Fig. S1. Combined efforts of qMS and cryo-EM to determine how SrmB acts in 50S ribosome

13

assembly. The protein levels report on the total amount of each protein relative to an internal

14

standard compared to an intact ribosome as an external standard (spike). Similarly, the qMS

15

levels report the relative amount and identity of rRNA modification levels relative to 23S

16

standard. CryoEM from selected fractions is then used to explore the structures of the 40S

17

ribosome assembly intermediates.

18 19

Fig. S2. Several biological replicate measurements were obtained using fractions spanning the

20

entire pre-50S : 50S : 70S regions of a gradient (Ex. 1 and 2 in WT and Ex.1 in ∆srmB ) or

21

across the pre-50S : 50S region (Ex. 3 and 4 in WT and Ex. 2 and3 in ∆srmB ), where the

22

largest changes in modification occupancy were found. The resulting data were merged for

23

hierarchical clustering analysis performed using Euclidian distance metric and average linkages.

24

Based on the dendograms, 23S modification were divided into three groups of early (red),

25

intermediate (green), and late (cyan) events during ribosome assembly in wild type and ∆srmB

26

cells.

26

1 2

Fig S3. C(2501) is partially modified in 23S of WT and ∆srmB. Cm(2498) was monitored via

3

RNase A fragment 2497-A(Cm)C-2499. RNase U2 fragments 2498-(Cm)CU (ho5C)G-2502 and

4

2498-(Cm)CU(C)G-2502 were used to profile ho5C (2501) and C(2501), known to be partially

5

modified. While (Cm)CU (ho5C)G-2502 is dominant, some quantities of 2498-(Cm)CU(C)G-

6

2502 are resolvable in both WT and ∆srmB , enabling us to roughly estimate fraction of 2501

7

modified in 70S ribosomes. (a) Representative spectra and their least-squares fits reporting on

8

the presence of Cm(2498), ho5C(2501), and C(2501) in sample 80 from ∆srmB. (b) 14N/15N

9

ratios were calculated for each of the four samples from 50S-70S region, with ∆srmB particles

10

being 14N labeled and 23S-WT being 15N labeled. After normalizing to the amount of

11

unmodified 23S present in the sample and the reference (e.g., 14N/15N = 1.38, sample 80), we

12

found that Cm(2498) is stoichiometrically present (14N/15N = 1.45 vs. 1.38), however

13

unmodified 2501 is 2.5 more abundant in ∆srmB, and ho5C(2501) is slightly substoichiometric.

14

Furthermore, we made two assumptions: first is that 2498 is 100% modified in both ∆srmB and

15

WT; second that ho5C(2501) and C(2501) fractions add up to 1. Using these assumptions,

16

fraction C(2501) hydroxylated were calculated for each of the four samples, and are shown

17

together with average and sd. The analysis suggests, that 50S and 70S particles in ∆srmB have

18

larger quantities of unmodified C(2501) than WT 70S.

19 20

Fig. S4. Unfiltered CryoEM maps. (a-c) Shown from left to right: front view, back view rotated

21

180°, Euler angle plot, and plots of global half-ma p 3DFSCs, three 3DFSC isosurfaces at a

22

cutoff of 0.5 in three axial orientations describe the isotropy (directional resolution) of the refined

23

maps. (a) SrmB-A class. (b) B class. (c) C class. (d) FSC curves with the colors of the curves

24

corresponding to (A-C). The resolutions of the classes are 5.7Å (Class A), 4.9Å (Class B), and

25

4.7Å (Class C). (e) Representative micrograph. (f) cryo-EM data analysis workflow.

26 27

1

Fig. S5. Assembly blocks from Fig. 4 shown on PDB ID 4YBB. (a) The full complement of

2

assembly blocks are shown on the top, followed by Block 5 (purple), Block 4 (blue), Block 3

3

(green), Block 2 (yellow), and Block 1 (red, not shown in Fig. 4). (b) Occupancy matrix for Group

4

1.

5 6

Fig. S6. (a) B class with non-native “bridge” highlighted in purple. (b) C class with non-native

7

“bridge” highlighted in purple.

8 9

Fig. S7. Relative levels of the SSU and LSU r-protein in ∆srmB vs WT measured using whole

10

cell proteomics. Red arrows indicate levels of S9 (a,b) and uL13 (c,d) that were found to be

11

significantly depleted in (Iost and Jain 2019), but they are not found to be significantly depleted

12

here. (a) Ratio of ∆srmB/WT SSU proteins at 37°C. (b) Ratio of ∆srmB/WT SSU proteins at

13

18°C. (c) Ratio of ∆srmB/WT LSU proteins at 37°C. (d) Ratio of ∆srmB/WT LSU proteins at

14

18°C.

28

a

CP L1 stalk

L7/12 stalk

c

1022-GUGGGAA-1028 (23S) uL25

e

1034-GUGGGAA-1040 (23S) uL13

b

Fig. 1. Anatomy of the 50S subunit (PDB ID 4YBB) including (a) the uL1 stalk (orange), CP (red), and L7/12 d stalk (yellow); (b) areas of contact between the 50S and 30S subunits uL24 nt200-400 (blue); (c) helices invovled in the peptidyl tranferase center (red, individual uL4 amino acids involved colored dark 25-UCCCAC-30 (5S) red) ; (d) the SrmB binding site (rRNA in dark green, proteins uL4 and uL24 in green and light green, respectively); and (e) mutations that can ameliorate ΔsrmB (orange) which occur near the region of uL13 (red) and uL25 (dark red) binding.

Group 2

Group 1

1.0

0.5

0.1

50S 40S

x

L22 L16 L6 L36 L35 L32 L9 L31 L7 L10 L30 L13 L33 L28 L27 L25 L14 L34 L11 L19 L2 L15 L5 L3 L17 L1 L18 L4 L23 L29 L24 L21 L20

Relative Protein Level

Group 3

Protein

Fig. 2. Relative quantification of r-proteins of 40S from ΔsrmB (pink) and of WT 50S ribosome particles (black). The relative 50S uL20 protein levels were normalized to 1, and remain consistent throughout the experiment. (Pink) Relative levels of proteins in the 40S intermediate. The proteins fall into three groups: less than 0.4 (gray), between 0.4 and 0.92 (medium gray), and greater than 0.92 (dark gray). If no peptides were found, the protein is represented by an x; otherwise, data was taken in triplicate, data from only one peptide are shown as an open circle, and data from more than one peptide are shown as closed circles.

a

b

WT

ΔsrmB

70S

70S

50S

50S

30S 40S

30S

1.2 1.2

Anna-WT-plot

29 30 31 32 33 34 35 36 37 38 39

1 745 6A(1618) m 1618 1835 1911 m2G(1835) 1939 2604 m 5C(1962) 2457 2552 2503 m6A(2030) 2501 2498 1962 m7G(2069) 2030

0.8 0.8

Early Intermediate Late

0.4 0.4 0.2 0.2 0 0.0

5

m G(745), Ψ(746), m U(747)

1 1.0

0.6 0.6

Fraction #

relative abundance of modifications

relative abundance of modifications

Fraction #

2069 Gm(2251) 2251

2

m A(2503), Ψ(2504) Ψ(2457), Ψ(2580)

5

m U(1939) Cm(2498)

5

ho C(2501)

28

30

32

34

36

38

Ψ(2604), Ψ(2605)

40

L36 Helix 89

Early Intermediate

0.6 0.6

1962

2

A(2503), Ψ(2504) m 2030 2069 Ψ(2457), 2251 Ψ(2580) Cm(2498)

0.2 0.2

0.00

5

2498 Gm(2251)

Late

0.4 0.4

5

m U(1939) Ψ(2604), Ψ(2605)

73

75

77

79

81

83

85

5

87

ho C(2501)

ho5C(2501)

Um(2552)

5S rRNA

L30

L16

3

Ψ(1911), m Ψ(1915), Ψ(1917)

C27

L25

L4 L13

L11

0.8 0.8

5S rRNA

C2501

1 6 1618 G(1835) m21835 1911 m51939 C(1962) 2604 A(2030) m62457 2552 2503 G(2069) m72501

m G(745), Ψ(746), m U(747) A(1618) m 745

Cm(2498)

Cm(2498)

C2498

73 74 75 76 77 78 79 80 81 82 83 84 85

1.01

3

Ψ(1911), m Ψ(1915), Ψ(1917) Um(2552)

ho5C(2501)

c

1.2 1.2

L4

180°

L4

C27 L25 L25

L30

90°

L21

L16

L11

L20

Fig. 3. Quantitative MS reveals the relative order of individual 23S rRNA modification events. (a) Sucrose density gradient profile of WT. Fractions are denoted by vertical grey lines. Relative rRNA modification levels were calculated across the sucrose gradient and normalized to the levels of unmodified 23S in each fraction. Modifications are classified as early (red), intermediate (green), or late (cyan). (b) Sucrose density gradient profile of ΔsrmB. Cm(2498) and ho5C(2501), which differ in order between WT and ΔsrmB strains, are labeled for each plot shown. (c) Relative position of C2498 and C2501 within the crystal structure of the bacterial ribosome. r-proteins are shown in grey. Helix 89 of the 23S rRNA, which contains C2498 (red spheres) and C2501(cyan spheres), is shown in yellow. Helices 41-44 of the 23S rRNA, which contain the sites of mutations that stabilize the growth defect at cold temperatures in ΔsrmB strain (A1039 and A1027) are in green. C27 is the site of another stabilizing mutation in the 5S rRNA (grey). Structure used was PDB ID 4YBB .

L11

Helix 41-44 L21

L6 A1039

A1027

L21

Helix 41-44 L6

L13 A1039

A1027

L20

L13

Helix 41-44

Helix 89 L6

ABC

c

Block 5

e

Block 4

Fig. 4. Analysis of the three 40S intermediate cryoEM structure. All cryoEM classes have been filtered to 10Å and are shown with a soft mask. (a) The least mature class: A class. (b) The intermediate class, B class. (c) The most mature class observed in the data, the C class. Blocks of structural elements in (a-c) are colored according to the occupancy analysis in d. (d) Hierarchical clustering heatmap of rRNA helix and r-protein occupancies in the three structures as referenced to PDB ID 4YBB. The largest Block 1 (Fig. S5) is not shown here, and has 100% occupancy across all structures. (e) Structural elements of the rRNA helices defined by the occupancy analysis mapped on to the 2D 23S rRNA domain structure. Colors correspond to the blocks in (d). Block 1 is shown in red. rRNA domain contacts that span across domains are shown in solid lines.

Block 3

b

d

Block 2

a

0

1

A Class

B Class

nt 200-400 L4

SrmB binding site L24

h42

C Class

4YBB nt 200-400 L4 L24

L25 L25

uL13 disruption

h42

L13

L13

5S h41

Suppressor Mutations

Non-native RNA contacts

h42

5S h41 h42

h97 h41

L20 L21

h41 L20 L21 h97

Fig. 5. Specific areas pertinent to SrmB binding in reference to the theoretical cryo-EM density of 4YBB. From left to right: the A class, B Class, C class, and the theoretical 4YBB EM representations, and the 4YBB model (right column) are represented in gray. The original, non-filtered cryoEM densities are used for visualization. The thresholds used for display were set to 3σ, which shows “dust” that is related to areas of compositional or structural heterogeneity and flexibility. Color scheme for each row is at the end of the row and in the first column. (Row 1) The SrmB binding site. L4 is colored in yellow green, L24 is colored in dark green, and nt200-400 are colored in sea green. (Row 2) Areas of known disruption in ΔsrmB. uL13 colored in red, bL25 in dark red, h42 is dark orange, and the black arrow points to density blocking uL13 binding. (Row 3) Sites of known SrmB deletion suppressor mutations. h42 is colored in light blue, h41 is in blue and the region of the 5S is colored in navy. (Row 4) Areas of non-native density that could be extensions of local rRNA helices. h41 is in blue, bL20 is in hot pink, bL21 is in dark purple, and h97 is in pink.

a b

*

*

**

**

** * * * * * * ** *

*

** * *

E K Q T G K P S K K V L A K R A E K K K A K E K E K P R V K K R H R D T K N I G K R R K P S G T G V P P Q TT E E Predicted minimum length: 112Å

Precursor

SrmB uL13

uL13

50S

Fig. 6. Potential pathways for SrmB to act in ribosome assembly. (a) Sequence of the C-terminal tail of SrmB. The blue box indicates predicted α-helix content, otherwise, the rest of the C-terminus is predicted to be uncoiled. Red stars indicate positively charged amino acids that could participate in RNA binding. The predicted minimum length of an α-helix, followed by random coils, has a minimum end-to-end length of 112Å, which is long enough to span the front of the ribosome from the uL1 stalk to the uL7/12 stalk. (b) SrmB could regulate the expression of uL13, which causes the ribosome assembly pathway to stall under cold-stress conditions (c) The C-terminal tail of SrmB could act as a folding chaperone to ensure proper central protuberance docking and rRNA folding around the uL13 binding site after SrmB docks into its binding site.(d) SrmB could stabilize the uL4:uL24:nt 200-400 interactions, thus providing a stable base that could prevent misfolding in further rRNA folding events. In this case, it is only the binding of SrmB that stabilizes the rRNA, and the C-terminal tail is not needed for further structural rearrangements. (e) SrmB could also act as a dock for other proteins, either by means of its C-terminal tail or by some other protein:protein interactions. In this case, SrmB would guide the rRNA into the proper conformations for docking and release of other proteins, such as methyltransferases (such as RlmM) or hydroxylases.

SrmB

c

50S

Precursor

SrmB

d

50S

Precursor

SrmB

e

Precursor

Protein Factors

50S

Highlights • SrmB deletion causes compositionally and conformationally diverse intermediates • Non-native density observed in the uL13 binding site and suppressor mutation sites • Defects in central protuberance, uL7/12 stalk, peptidyltransferase center • SrmB deletion causes reordering of rRNA modifications • SrmB might reroute stalled complexes with structural defects