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1 SUPPLEMENTARY INFORMATION doi: /nature18647 Contents 1. Introduction 1a. Overview of eukaryotic translation initiation p. 2 1b. Ribosomal scanning of mrna p. 3 1c. Recognition of start codons by small ribosomal subunits p Results and Discussion 2a. Overview of the Translation Complex Profile sequencing (TCP-seq) technique p. 8 2b. Ribosomal footprints on mrna detected by the TCP-seq method p. 9 2c. Impediments to scanning of mrna p. 11 References p

2 RESEARCH SUPPLEMENTARY INFORMATION 1. Introduction 1a. Overview of eukaryotic translation initiation Translation in eukaryotes is a highly regulated process involving a plethora of auxiliary factors, which collectively influence the rate of translation both of individual mrnas and the entire transcriptome. Translation initiation serves as an integration point for regulatory inputs. Key steps of translation initiation are considered to be important subjects of control mechanisms delivered by translation initiation factors and their interactions with other regulators reviewed in, e.g. 2-5, These steps include the recruitment of the small ribosomal subunit (SSU) as a preinitiation complex (the 43S complex; eif1:eif1a:eif2:gtp:met-trnai Met :SSU:eIF3:eIF5) to the 5' end of the mrna, advancement of the SSU in the 3' direction of the 5' UTR (termed scanning by M. Kozak 8 ) until the complex encounters and recognizes an initiation codon, and subsequent conformational changes leading to large ribosomal subunit (LSU) joining and the onset of translation (Extended Data Fig. 1a). Additionally, intrinsic features of the mrna can interact with initiation processes and provide another layer of translation regulation, individually for each transcript. Specifically, untranslated regions of mrna can influence translation efficiency both positively and negatively. Well-known examples of negative regulation in 5' UTRs include short upstream open reading frames (uorfs) 52 that act as decoys for initiating ribosomes, or protein binding elements such as the iron responsive element (IRE) 53 that are stabilized by their ligands and intercept initiating SSU. Heavily structured or excessively short 5' UTRs can also contribute to downregulation of protein synthesis from particular mrnas Positive regulation by 5' UTR sequences is implemented by occurrence of internal ribosome entry sites (IRESs) that promote initiation without requiring the main translation initiation factors and can be additionally augmented by IRES trans-acting factors (ITAFs) 58-60, incorporation of special regions that can directly attach initiating ribosomes 18,61, and classical examples of secondary structures downstream of the initiation codon that are thought to slow down initiating SSU over the start sites and thus decrease the probability of start codon bypass 62. As a stand-alone regulator, nucleotide sequence around initiation codon (termed nucleotide context by M. Kozak 63,64 ) is a special feature of eukaryotic initiation that is considered as one of the most important intrinsic regulators defining general efficiency of translation from a given mrna. Elements in the 3' UTRs are also able to interfere with translation, primarily by affecting the early stages of translation initiation, such as SSU attachment to cap structures on the mrna 5' ends 65. Mechanisms of eukaryotic translation regulation are analysed by either classical methods where individual mrnas are assayed in detail with in vitro biochemical and structural experiments or transfection of reporter constructs into cells, or by the ribosomal (translation) profiling experiments 6,66-74 where occupancy of open reading frames by ribosomes is analysed across the transcriptome directly from live cells. Lack of methods to capture translational complexes beyond the elongation stage of translation in a steady-state in vivo environment prompted us to create the translation complex profile sequencing (TCP-seq) method. 2

3 SUPPLEMENTARY INFORMATION RESEARCH 1b. Ribosomal scanning of mrna Scanning of mrna by the SSU was proposed almost 40 years ago by M. Kozak in order to fit the initiation model to the predominantly monocistronic nature of eukaryotic mrnas, the universal requirement of a 5' cap structure on mrnas for efficient translation initiation, and the rarity of AUG codons in mrna 5' UTRs 8. The scanning model was later extended to incorporate the effect of the sequence surrounding the start codon on the frequency of its recognition This leaky scanning model acknowledged the partial bypass of some AUG codons observed in cases where the codon s nucleotide context is not optimal. Additionally, non-aug near-cognate codons can be recognised as start sites, especially if they are found in a strong nucleotide context 79,80. Preferences for the strength of the nucleotide context and recognition of near-cognate start codons vary to some degree across eukaryotes. In Saccharomyces cerevisiae the optimal nucleotide context reportedly is AAAAUGG 81, and CUG, UUG/GUG are the most efficient near-cognate start codons 80,82. Most intriguingly, the scanning model of mrna has not yet been supported by any direct evidence: SSU in transit to the start codons have not been isolated biochemically, or trapped attached to their mrnas during naturally occurring translation 2,5,83,84. This is likely a consequence of the highly dynamic nature of scanning SSU and the unavailability of drugs that could specifically stall them on mrna without detachment. Key biochemical observations that support scanning in the form of a linear 5' 3' directed motion (as originally proposed by Kozak) include the first AUG rule where the most upstream (5'-end proximal) strong start codon is normally selected as cognate ORF start 85, the negative effect of stable RNA structure in front of the start site on efficiency of translation, and the positive effect if such structure is located immediately after the start site, presumably slowing down the motion of the scanning SSU and thus increasing the probability of start codon recognition 57,62,86,87. Additional indirect evidence is provided by the linear dependence of the initiation time on the length of the 5' UTR observed in both yeast and higher eukaryotic in vitro translation systems 88. One of the strongest indications for Kozak s scanning is the detection of the so-called complex I between initiating SSU and rabbit β-globin mrna by the toeprinting assay in an in vitro reconstituted translation initiation system 89,90. This complex formed upstream on mrna (proximal to its 5' end) when eif1 and eif1a were omitted, and was prevented from forming or converted into a normal start codon initiation complex located downstream on mrna when these factors were present in, or subsequently added to, the reaction mixture 89. However, perhaps the best evidence for the scanning SSU until now remains the observation of faster sedimenting TCs using in vitro translation reactions where recognition of the start codon was blocked by antibiotic edeine 13,91. It was also reported that mrna fragments protected from RNase digestion in this fraction have higher complexity of sequence than the 5' UTR regions of mrna alone 13, however, methodology to recover the exact positional data of the SSU locations was not available at that time. Other mechanisms for start codon searching have been proposed 9, and some are in principle compatible with most of the existing biochemical evidence. The mrna looping hypothesis 9,102,103 is a major alternative to linear 5' 3' directed motion, conjugating earlier concepts of ribosomal shunting and maintenance of cap attachment ( cap-tethering ) during translation initiation. According to this model, start sites on mrna are selected as a result of a series of SSU hops between the cap structure of mrna where it initially docks, and a number 3

4 RESEARCH SUPPLEMENTARY INFORMATION of landing sites that have higher affinity and/or structural availability for the initiating SSU 9 (Extended Data Fig. 1b). It has been proposed that such a search mechanism can result in the prevalent initiation on the most 5'-end proximal strong start site and comply with the observed dependence of the initiation time on 5' UTR length 9. However, in contrast to the canonical linear 5' 3' directed motion model, mrna looping is less consistent with a cap-severed mode of initiation (as it allows only one SSU to initiate over 5' UTR at a given time), and would result in clustering of the SSU on some particular sites of the 5' UTR that would accompany efficient translation, as well as in the increased SSU presence over the 5' UTR regions immediately adjacent to the mrna 5' ends. According to current knowledge, the SSU is bound with at least eifs 1, 1A, 2:GTP:Met-tRNAi Met, 3 and 5 when it initially docks at the 5' cap structure of mrna 2,84 (Extended Data Fig. 1a). The docking involves interaction of eif3 as part of the initiating SSU with eif4g as part of the cytoplasmic cap-binding complex eif4f (eif4a:eif4e:eif4g) that attaches to the cap structure of mrnas. It is hypothesised that the initial eif4f attachment to the mrna cap creates a structurally accessible region of the 5' UTR nearby, via the RNAhelicase activity of, presumably, eif4a, and thus facilitates loading of the initiating SSU on the mrna. The exact molecular mechanism and sequence of events during SSU loading on mrna remain unknown, and most importantly, it remains unclear whether the initiating SSU, upon departure from the mrna 5' end, preserves the contact with the cap structure until start codon recognition ( cap-tethered scanning; Extended Data Fig. 1c lower model), or loses it while still in transit ( cap-severed scanning; Extended Data Fig. 1c upper model), allowing multiple entries of SSU into the same 5' UTR 14,88,104. However, it is well established that effective scanning of mrna additionally requires presence of free (not complexed in eif4f) eif4a, single-stranded RNA binding proteins eif4b/eif4h, and hydrolysis of ATP. In the most recent observations, it was shown that a minimal combination of the central domain of eif4g, eif4a and eif4b/eif4h can result in a processive ATP-dependent RNA-helicase that is specific to the RNA strand direction 105 and therefore may constitute a core component of the scanning machinery. Notably, to perform scanning, the SSU has been shown to adopt a special conformation, interacting with the template mrna in a way that is supposed to facilitate linear diffusion 19,21,24,106,107. This is thought to be achieved by maintaining a minimal tight contact area between the mrna-binding cleft of the initiating SSU and mrna. The head of the SSU ( the latch for mrna) is repositioned in the scanning complex in a way that renders the mrna-binding channel wider, fully exposing the mrna at the entry (3'-ward on mrna) side of the SSU, including the SSU A-site area. This is complemented by the absence of a stable structure of the eif1a-n-terminal part over the SSU A-site and the so-called POUT (or scanning P/I 19 ) configuration of the eif2:gdp-pi:met-trnai Met complex and initiation factors eif1 and eif1a, where initiator trna is positioned outward from the SSU compared to its elongation P-site position and is angled towards the SSU E-site, with eif1 blocking the trna from fully entering the SSU P-site (see Scanning schematic in Extended Data Fig. 10b for a summary model). Because eif2 is known to make contacts with both head and body of the scanning SSU, the eif2:gdp-pi:met-trnai Met complex and eifs 1 and 1A create a relatively loose window in the region of the SSU P-site, through which a single-stranded mrna chain can be threaded and inspected for codon-anticodon interactions by Met- 4

5 SUPPLEMENTARY INFORMATION RESEARCH trnai Met[19]. While the linear sliding of single-stranded mrna is allowed, the interaction of the scanning SSU and mrna is strong and intimate enough for scanning to be processive 88 and recognition of the start codons to be highly efficient, as can be observed by the rarity of leaky scanning through strong initiation sites 108. Interestingly, biochemical data supports that a minimal set of eifs 1, 1A, 2 (and 3 for some cases) and Met-tRNAi Met is sufficient to create an SSU capable of mrna inspection for cognate start sites in the absence of ATP hydrolysis (presumably by free, non-directed sliding over the mrna chain), provided that the 5' UTR is devoid of strong secondary structure 55,61. Although the configuration of the mrna, Met-tRNAi Met and the mrna binding cleft are well established for the scanning SSU, highly detailed positions of eif3 and eif4g (or eif4f) are not known. It is therefore not clear what mechanism provides for directionality of the scanning 83, however, two key alternatives can be highlighted 104,109. In one case, the scanning factors, and notably eifs 4A, 4B, 4G known to be capable of unwinding RNA structures, would reside (i.e. make tight contacts with mrna) on the leading edge of the scanning complex (and thus towards the 3' mrna end relative to the SSU; Extended Data Fig. 1d lower schematic) and would move the SSU 3'-ward, by pulling it through the unwound mrna 109. In the other case, the scanning SSU would be pushed forward (or its rollback would not be allowed 15 ) by these factors located at the trailing edge of the complex (towards the mrna 5' end relative to SSU; Extended Data Fig. 1d upper schematic), with SSU acting as an integral part of the RNA helicase in this scenario 14. An unlikely yet possible situation could be that most stable SSU contacts extend towards the mrna 5' end by the scanning factors, whilst they perform their action on the 3', incoming portion of the mrna. In this arrangement, it would require mrna to make almost a complete loop around the scanning SSU to support the pulled SSU model. There is some existing structural evidence to support the pushed-ssu arrangement because eif4g is thought to be located on the solvent side of the SSU near the mrna exit site 14. This is further supplemented by the SSU solvent-side location of most of the eif3 mass (although some eif3 domains can reach the intersubunit SSU surface 19 as well) 14,110. Finally, in the original works where protection of mrna fragments by initiating SSU was studied in vitro, it was concluded that scanning SSU can possess 5'-extended footprints 111,112. Additional RNA helicases may assist translation initiation and can stably associate with scanning SSU 67,88, In the mammalian system, it was shown that RNA helicase DHX29 can reside at the mrna entry site of scanning SSU 110,116, which confers on the SSU the capacity to initiate on mrnas with highly structured 5' UTRs in vitro 114. However, DHX29 is less abundant than eif4a 117 and is considered to assist the regular scanning process through the rearrangement of mrna position on the SSU and/or the SSU RNA-binding cleft itself, resulting in (at least partial) bypass of the high-energy structures by the scanning SSU 118. The exact layout of the full scanning complex on mrna is not established and the existing structural studies are based on partial reconstitutions of the scanning SSU in vitro. Because the start codon location is one of the main effectors of transcript-specific control of protein synthesis in eukaryotes, elucidating the mechanism of ribosomal scanning of mrna and establishing methods to assay this process across the transcripts of living cells are of great interest. 5

6 RESEARCH SUPPLEMENTARY INFORMATION 1c. Recognition of start codons by small ribosomal subunits Recognition of the start codons by initiating SSU in eukaryotes is a well-understood process reviewed in, e.g.2,3,47, (see Extended Data Fig. 1a for overview schematic). The most stable intermediates of this process can be isolated biochemically or assayed by primer extension assays ( toeprinting ) on mrnas 122, and three-dimensional structure reconstitutions exist for many of the start codon recognition complexes assembled from purified components in vitro. The process requires three major stages to complete before an elongation-capable (80S) ribosome is assembled on the start codon. First, the initiator trna within the scanning SSU forms Watson-Crick interactions with a start codon positioned in the scanning SSU P/I ( POUT') site that may stall scanning SSU movement along the 5' UTR 19,21,106,123 (see Start codon pausing schematic in Extended Data Fig. 10b for a summary model). Key 3 and +4 residues of the start site nucleotide context are proposed to slow down the scanning SSU over the initiation codon resulting in high efficiency of recognition and low leaky scanning rate, and additionally stabilise codon-anticodon interactions upon their establishment primarily through contacts with ribosomal RNA and eif2 21,124. It was proposed that eif2-bound GTP hydrolysis may occur during scanning, and when the SSU initially pauses over the start codons, eif2 can be in its GDP-bound form; however the Pi product is not yet released 20. Next, as a result of eif1 displacement from the ribosomal P-site by stable codonanticodon interactions, an induced-fit structural change to a closed conformation occurs whereby initiator trna is moved inwards along the SSU mrna-binding cleft and fully occupies the SSU P-site ( PIN conformation) 19,21,106 (see Start codon recognition schematic in Extended Data Fig. 10b for a summary model). This triggers a major rearrangement of the initiating SSU and changes the positions of eifs 1A, 2 and 5, resulting in rotation of the SSU head, narrowing of the SSU mrna-binding cleft, closing of the latch at the SSU mrna entry site and formation of a stable structure by the N-terminal portion of eif1a over the SSU A-site 19,106. This rearrangement is thought to increase tight contacts of mrna with the initiating SSU and prevent further SSU sliding on mrna, fixing it in frame with the trnabound start codon 19,21,106. Upon this rearrangement, Pi is released from eif2-gdp, weakening its affinity to initiator trna and SSU, and eif1 is completely ejected from the initiation complex 20,80,125. Finally, eif5b:gtp displaces eif2:gdp (perhaps together with eif5) from the initiation complex and binds the initiator trna 126,127 (see Pre-subunit joining schematic in Extended Data Fig. 10b for a summary model). This incurs repositioning of the initiator trna by moving its acceptor stem closer to the centre of the SSU body so that it acquires conformation similar to that of elongation P-site trna 23,24. At this stage, the eukaryotic initiation complex closely resembles prokaryotic initiation where IF1 and IF2 have activities analogous to eif1a and eif5b, respectively 128,129. Analogous to prokaryotes, binding of eif5b occurs from the mrna entry side, connecting the acceptor stem of initiator trna with the SSU body below the mrna-binding platform. This renders the initiation complex capable of LSU attachment and an elongation-competent initiation ribosome located at the start codon is formed. LSU joining further induces hydrolysis of eif5b-bound GTP and eif5b:gdp and eif1a dissociation from the complex 127,

7 SUPPLEMENTARY INFORMATION RESEARCH Interestingly, while the steps of start codon recognition are very well characterised in vitro, and accurate kinetic estimates exist for timings of POUT to PIN transition, Pi release and dissociation of eif1 125, there is not much knowledge about dynamics of start codon recognition in live cells. This knowledge can be particularly important because different transcripts might have differential influences on the rates of each start codon recognition step though local mrna sequence (and structure), resulting in individualised control of this process for each species of mrna. 7

8 RESEARCH SUPPLEMENTARY INFORMATION 2. Results and Discussion 2a. Overview of the Translation Complex Profile sequencing (TCP-seq) technique While cycloheximide or other elongation blocking compounds such as lactimidomycin, anisomycin, etc. are commonly used to freeze translating ribosomes in place, no equivalent small-molecule inhibitor of scanning has been found. However, it has been recently reported that rapid fixation of live cells with 1 to 3 % formaldehyde at low temperatures preserves SSU-mRNA complexes as observed by sucrose gradient fractionation 10. The resultant sedimentation profiles were demonstrated to be indistinguishable from profiles where ribosomes were stalled on mrnas by cycloheximide, except that an elevated half-mer peak appearance was observed and attributed to the presence of additional SSU fixed on mrna together with complete ribosomes 10. The same approach has been successfully used to trap ribosomes at different maturation stages 131, as well as to analyse distribution of the mrna-protein interactions upon circularization of mrnas during translation 33. Therefore, we elected to employ in vivo formaldehyde crosslinking to immobilise initiating SSU on mrna. Given that estimated scanning speed in yeast translation initiation is about 10 nt/s (at 25 C) 88 and the typical formaldehyde reactivity half-time in vivo is ~2.5 seconds (at room temperature) 132, we expect that mrnas with 5' UTRs longer than 25 nucleotides will show scanning SSU distribution after fixation reflecting that in live cells. The actual resolution may be significantly better than this as formaldehyde reaction rates with organic amines increase at lower temperatures 133 while translation slows down 134,135, however, unambiguous discrimination of 5' UTR SSU footprints from start codon SSU footprints is constrained for mrnas with short 5' UTRs. Translation initiation complexes assembled on start codons, such as start codon PIN SSU, will likely be captured with minimal distortion as they are relatively long-lived, with typical overall times of complex conversion upon recognition of the start codon in yeast being in the order of 2.5 seconds 125. One limitation of the TCP-seq as developed by us is that only mrnas with at least one translating ribosome attached will be sedimented in the first ultracentrifugation step, and thus mrnas that are completely non-translated (but which may have SSU subunits attached) will not be observed using this technique. We introduced this sedimentation step to avoid overwhelming the RNA fragment library with SSU rrna from the likely abundant free SSU in the cytoplasm, as well as to focus initially on highly translated, active mrnas. Other methods for purifying mrnps from free cytoplasmic SSU (e.g. immunoprecipitation by mrna-bound poly-a binding protein, etc.) may be employed to access completely untranslated targets, or alternatively use of efficient techniques for ridding the highly fragmented RNA samples of rrna fragments could circumvent this requirement. Here we prioritized fidelity of the original RNA fragment population and thus did not employ stringent size-selection that reduced the complexity of the population of contaminating rrna fragments in the conventional ribosomal profiling technique, thus facilitating their removal 136,137. We also did not deploy rrna removal at the RNA-level and instead used the cdna-based PDD method 34,35, which, while only moderately boosting non-rrna representation (due to the small modal fragment size of the library), does not carry the risk of creating new fragment ends. 8

9 SUPPLEMENTARY INFORMATION RESEARCH Nonetheless, these types of alterations to the TCP-seq technique might be employed if the trade-off between mrna content yield and footprint size fidelity can be relaxed, depending on the particular experimental goals. In particular, as we now know that scanning SSU generally produce footprints mostly longer than ~19 nt but shorter than ~75 nt, a size-selection step with a stringent upper and lower bound on footprint size could be employed if analysis of scanning SSU were the sole aim. As currently applied by us, TCP-seq libraries did not exhibit significant levels of PCR duplication, offering the potential of at least 10-fold deeper sequencing, or 10- fold reduction in the starting material, while keeping the proportion of library duplicates still in an acceptable range for the ribosome fraction. 2b. Ribosomal footprints on mrna detected by the TCP-seq method Because TCP-seq is universal to any type of translation complex on mrna, we were able to assess distribution of the SSU and the elongating ribosomes in the same sample. Multiple datasets for ribosome occupancy on yeast mrnas are available, including the original ribosomal (translation) profiling experiment 136 and more recent studies 43. This allowed us to confirm the validity of our approach further by comparing the footprints positions recorded from our TCP-seq ribosome fraction to the previous observations with ribosomal profiling. In contrast to the total input (Fig. 1a) fraction where 5' ends of reads map diffusely across the entire length of transcripts, footprint 5' ends of ribosome footprints obtained with TCP-seq concentrate mostly within the limits of the ORFs (Extended Data Fig. 2a), with little overall heterogeneity in length (Fig. 2a,b; Extended Data Fig. 2d,e). There is minimal presence of ribosomes on the 5' and 3' UTRs, however, a small peak of appropriate ribosome footprint length indicates some translation of upstream ORFs (uorfs) and perhaps a very minor intrusion of ribosomes into the 3' UTR (Extended Data Fig. 2c,d-f). These observations are generally consistent with prior ribosome profiling data, however, we detect only slight accumulation of ribosome occupancy at the first codon (Fig. 2a; Extended Data Fig. 2a, 3a, 5a,c, 6a,c) indicative of fast, non-limiting commencement of elongation. There is also a slight trough in the ribosome footprints in the last five sense codons of mrnas, possibly resulting from partial inclusion of these complexes into heavier structures that sediment differently, or from increased translation speed (Fig. 2b; Extended Data Fig. 3a, 5b,d, 6b,d). A higher frequency of ribosome footprints was reported over start codons when ribosomal profiling is performed with the use of elongation-blocking compounds 66,69,136. This feature of ribosome profiling is specifically used to highlight initiation sites on mrna when ribosomes with a single trna in the P-site are stalled by lactimidomycin, while ribosomes fully engaged in elongation are disassembled through abortive reaction with puromycin 69. Increased accumulation of ribosome in 3' UTRs 68 may also in part result from cumulative deposition of elongation-arrested ribosomes over some period of time when cellular translation components are still active. Because ribosome distribution over mrna is fixed near its in vivo steady-state during TCP-seq, this method can arguably be expected to generate a representation of ribosome footprints along mrnas that is less affected by residual translational activity during generation of ribosome footprints than conventional ribosomal profiling. Except for the minor proportions of footprints located at start codons, the ribosome footprints demonstrate an invariant length profile over the ORF regions and show a major modal footprint size of 31 nt, with a much less abundant 21 nt subpopulation (Extended Data 9

10 RESEARCH SUPPLEMENTARY INFORMATION Fig. 2e). Start codon ribosome size distribution is almost identical, although the modal footprint size was apparently slightly longer (32 nt) possibly reflecting a different configuration compared to elongating ribosomes (Fig. 2a; Extended Data Fig. 2e). Importantly, the density of ribosome footprints shows a 3-nt periodicity (Extended Data Fig. 3e,f) reflective of codonby-codon ratcheting of the ribosomes through mrna during active elongation. SSU footprints over ORF regions also demonstrate such periodicity (Extended Data Fig. 3e,f) and therefore a large proportion of them originate from elongating ribosomes that dissociated into subunits before fractionation, leaving the SSU attached to mrna. The overwhelming abundance of ribosome complexes relative to SSU (Fig. 1b) means that disassociation of only a tiny fraction of ribosomes would lead to significant presence of their SSU in the SSU fraction, as observed. When cycloheximide is used to stall ribosomes in the pre-translocation state in ribosomal profiling experiments, a homogeneous nt ribosome footprint is observed all across the ORFs 66,136 with 12/+15 nt of the footprint 5' ends and footprint 3' ends extensions (from 0 in the ribosome P-site). Ribosome footprint extensions from TCP-seq are similar with a 13/+17 signature (Extended Data Fig. 3a,b, 5c,d). This marginally longer modal ribosome footprint length and footprint extensions in our case could be a result of less stringent RNase digestion conditions. However, it is more likely that the longer ribosome footprints generated by TCP-seq are a consequence of formaldehyde crosslinking being able to preserve a more heterogeneous ribosome population. Unlike ribosomal profiling experiments using elongationblocking agents, TCP-seq does not force all elongating ribosomes into one particular sub-step of the elongation cycle, thus allowing capture of the diversity of elongating ribosome conformations. It has been previously demonstrated that ribosomal profiling performed without an elongation blockade detects a minor population of shorter nt ribosome footprints 66 and our observation of 21 nt ribosome footprints at low abundance is consistent with these data. Interestingly, the ribosome conformation resulting in shorter footprints was found to be stabilised by anisomycin, which presumably blocks ribosome at its post-translocation, rotated state 66. The protection of a shorter stretch of incoming mrna (on the ribosome's mrna entry side) has been proposed to be the basis of the observed shorter ribosome footprints in this case 66. Similarly, here ribosomes affixed at the start codons generate a subpopulation of 21 nt footprints also shortened at the 3' end to +7 nt, while footprint 5' ends are invariant at 13 nt in all cases (Fig. 2a; Extended Data Fig. 2e, 3a). The higher flexibility of the ribosome entry site than that of the exit site during elongation is reflected by a more complex pattern of footprint 3' ends fluctuations generated by TCP-seq (Extended Data Fig. 3a,b, 5c,d) and a broader distribution of ribosome footprint 3' ends over ORF codon registers compared to footprint 5' ends. When footprints are separated by footprint 5' ends into the three codon registers relative to the ORF, they exhibit a clear-cut monomodal footprint length distribution in the nt range, as well as a different overall abundance, for each of the three ORF codon registers. By contrast, attempting to use footprint 3' ends to classify ribosomes by register yields two footprint length maxima in footprint 3' registers 0 and 1 (Extended Data Fig. 3e,f). These maxima are separated by 3 nt, likely indicating that they result from pooling ribosomes from adjacent codons due to the wide variability of the extent of mrna protected at the ribosome entry site. This variability might result from minor alterations at the ribosome mrna entry channel during translation and 10

11 SUPPLEMENTARY INFORMATION RESEARCH explain the slightly longer modal ribosome footprint size detected with TCP-seq. Overall, ribosome footprint data obtained by TCP-seq are in good agreement with previous observations utilizing the ribosome (translation) profiling approach (also correlating with ribosome abundance recorded for individual mrnas, see Extended Data Fig. 3c), however, a more diverse range of ribosome states (and ribosome footprint lengths) is recorded by TCP-seq as a result of the formaldehyde crosslinking used to capture steady-state snapshots of translation. 2c. Impediments to scanning of mrna A key advantage of the TCP-seq method is its capability to record positions of the scanning SSU at steady-state. It is therefore possible to provide an assessment of whether scanning SSU distribute homogeneously or are clustered within the 5' UTR of particular mrnas (as can be assessed through our web interface to the TCP-seq data, Further, by analysis of the ratio of start codon SSU to 5' UTR SSU, a relative influence of scanning on translation efficiency can be determined and mrnas specifically regulated during the scanning stage of translation initiation can be identified. In the metagene analysis, transcripts with sufficiently long annotated 5' UTRs 45 (>75 nt) collectively show an upstream density of scanning SSU footprints per nt in a window from 70 nt to 50 nt, while there are up to 18,997 SSU footprints on the start codons in the same transcripts (with 5' end between 10 nt and 16 nt, subtracting background density), representing a ~43.5-fold higher density at the start codon than in upstream regions (start codon density calculated by condensing all start codon footprints onto a single nucleotide). This ratio is a maximal bound that assumes 5' UTR regions do not have introns or length heterogeneity, and is broadly in line with in vitro studies showing that per-nucleotide scanning 88,90 is approximately 25 times quicker than start-codon clearance 125. Inspection of individual mrnas with smooth progression of scanning SSU confirms this: SSC1 mrna, possessing 5' UTR-tostart codon peak SSU footprint coverage ratio in the lower rankings band (Fig. 3c), has a 5' UTR-to-start codon SSU footprint density proportion of ~1:25, as calculated using a central region of its 5' UTR (Fig. 3a, Extended Data Fig. 7b). It should be noted, however, that SSU footprints counts might not be strictly proportional between 5' UTR and start codon SSU as these complexes may be captured unequally due to theoretically possible differences in crosslinking efficiency. While the latter might skew the estimation of the scanning SSU-tostart codon SSU density ratio in absolute terms, comparisons of equivalent SSU complex subtypes between genes or between conditions would remain unaffected. In accordance with the homogeneous metagene distribution of the scanning SSU over 5' UTRs (Fig. 2a,c; Extended Data Fig. 2b), many yeast mrnas demonstrate smooth SSU footprint distribution across the entire 5' UTR, with low 5' UTR footprint density compared to the frequency of the start-codon associated SSU footprints (Extended Data Fig. 7). These mrnas include well-translated and ubiquitously present transcripts such as TDH1, ACT1 and SSC1 that are often used as transcription efficiency controls, as well as other mrnas with a 5' UTR length range up to about 200 nt (Fig. 3; Extended Data Fig. 7 for examples; most of mrnas from the lower band of Fig. 3c). The SYO1-TAP line demonstrates some decrease of the scanning SSU coverage in the 5' UTRs of such mrnas, with greater dominance of SSU footprints at start codons, as can be expected for a line with a mild large subunit deficit. 11

12 RESEARCH SUPPLEMENTARY INFORMATION Interestingly, many of these mrnas also demonstrate appearance of a peak of footprint 5' ends at the 30 nt position in start codon associated SSU footprints (e.g. ACT1, YAL005C, YLR208W in Extended Data Fig. 7), raising the possibility of queuing of a second scanning SSU behind start codon affixed SSU (see Fig. 4b; Extended Data Fig. 10a for a schematic) for rapidly scanned individual mrnas, that extends protection of mrna further 5'-ward by ~19 nt from the SSU stalled at the start codon (although a conformational change of the mrna exit channel of some initiating SSU is also a possible explanation). A similar effect of footprint length extension was previously described for stacking of elongating ribosomes 138. These 30 nt footprint 5' ends tend to decrease in the SYO1-TAP SSU library consistent with the overall lesser abundance of the scanning SSU in this strain. Of note, the major +6, +16 and +24 nt footprint 3' ends modalities reflective of the SSU conformational changes during start codon recognition (Fig. 4a, Extended Data Fig. 5c) revealed on the metagene level are all present within individual mrnas (e.g., ACT1, YPL131W, TDH3, YGL123W, YDL014W, SSC1, YGR240C mrnas in Extended Data Fig. 7). However, the frequency of these different start codon footprint 3' ends varies between mrna of each type, and this variance is well reproduced between the wild-type and SYO1-TAP strains (Extended Data Fig. 7, 9d-f). It is tempting to speculate that some local structural or sequence features of mrnas might affect the speed of different start codon recognition sub-steps (although sequence-specific bias in the RNA-seq libraries may also play a role). For instance, YAL005C and YLR20W mrnas are almost completely devoid of the shortest +6 footprint 3' ends at their start codons indicative of very fast transition from the initial pausing over the start codon during scanning to the PIN closed configuration of the SSU (Extended Data Fig. 7b). In summary, for all these mrnas recognition of the start codon appears to be more important than scanning in terms of speed of SSU complex processing, under conditions of exponential growth. Contrasting with the above, a population of mrnas exists that under the same conditions shows sites of high SSU footprint accumulation in the 5' UTRs (Extended Data Fig. 8; mrnas from the upper band in Fig. 3c; Supplementary Table 2). This is not due to library construction or sequencing artefacts as demonstrated by the relatively even coverage of reads from the input fraction and the lack of strong end-sequence bias detectable using MART (multiple additive regression trees; as implemented in the mseq R package 139 ) and other algorithms (Extended Data Fig. 4). An additional speculation could be that some of these footprints (as well as footprints in the 5' UTRs of other mrnas) derive from protection of mrna by some RNA-binding proteins, rather than from scanning SSU. Although a small proportion of such protected fragments could be present in our SSU libraries, this is highly unlikely because the number of these 5' UTR reads (adjusted to the overall library size) is decreased in the SYO1-TAP strain, confirming their relation to the availability of the SSU, and the fractions of mrna protected by SSU were co-purified with them by sedimentation velocity after RNase digestion, excluding the possibility of library contamination by footprints of RNAbinding proteins not associated with SSU. mrnas with longer 5' UTRs tend to exhibit impeded scanning more frequently, and almost every yeast mrna with a 5' UTR longer than 200 nt lacks fully homogeneous distribution of the scanning SSU footprints. mrnas with shorter 5' UTRs can also accumulate clusters of 5' UTR SSU indicating that this discontinuity is not a simple consequence of the 5' UTR length (Fig. 3c; Supplementary Table 2). Not all mrnas with unequal distribution of 12

13 SUPPLEMENTARY INFORMATION RESEARCH the scanning SSU over 5' UTRs can be considered important for regulation of translation initiation. If footprints from start codon SSU are more numerous than those from any one 5' UTR cluster, clearance of the start codons could still be the limiting step of initiation. To estimate the likelihood of translation regulation through impediments to scanning, we scored yeast mrnas by the peak coverage of the SSU footprints in the 5' UTR, normalized to the peak coverage of SSU footprints at the start codon of the main ORF (Fig. 3c; Supplementary Table 2). Hypothetically, clustering of the SSU in the 5' UTRs could be solely a consequence of translation initiation at uorfs or uaugs, features readily elucidated by conventional ribosomal profiling 140. mrnas known to possess functional uorfs (both with AUG and non- AUG near-cognate start sites) are indeed detected by TCP-seq with a high 5' UTR-to-start codon peak coverage ratio (Supplementary Table 2). Accumulation of SSU footprints in the 5' UTR is prominent for a classical example of translation regulation by short uorfs: the GCN4 mrna (Fig. 3b; Extended Data Fig. 8a). In seminal studies this mrna has been shown to act as a sensor for the cytoplasmic levels of eif2:met-trnai Met :GTP, reducing the number of SSU that can reach the start codon of the main ORF Under normal conditions of growth when eif2:met-trnai Met :GTP is abundant, special elements in uorfs 1 and 2 are proposed to permit resumption of SSU scanning after termination, however, these SSU reacquire eif2:met-trnai Met :GTP from the cytoplasm, terminate and dissociate from mrna upon translating uorfs 3 and In case of eif2:met-trnai Met :GTP deficiency, these SSU are thought to pass over uorfs 2-4 before eif2:met-trnai Met :GTP is rebound and they can thereby reach the start codon of the main ORF ,146,147. Ribosome footprints over uorf 1 are well detected by ribosome profiling experiments, there also is some evidence towards minimal translation of the uorfs 2-4, and ribosome presence in potential non-aug nauorfs 1 and 2 located further upstream that is increased upon starvation together with ribosome occupancy of the main ORF 136. Translation from the nauorfs has recently been shown to be dispensable for GCN4 control 148. TCP-seq captures an SSU distribution on the GCN4 5' UTR that is consistent with the known findings, however, more detail is provided. Start codon recognition on uorf 2 is better visualised and there is minimal detection of SSU footprints over uorfs 3 and 4, evidencing the preferential translation of uorfs 1 and 2 under normal growth conditions (Fig. 3b; Extended Data Fig. 8a). Notably, while SSU presence over uorf1 is increased in the SYO1-TAP line (repeating behaviour of the main start sites of the other mrnas in this strain), initiation on the uorf 2 is further depressed, although uorf 2 contains an AUG codon in the strong nucleotide context as well. This suggests that initiation on the uorf 2 is indeed performed by the SSU deriving from ribosomes terminated after translation of uorf 1 because translation of uorf 1 is repressed in the SYO1-TAP line due to the longer LSU waiting times. This excludes a possibility of internal attachment of SSU to uorf 2 or its translation by efficient leaky scanning of the uorf 1. Interestingly, while TCP-seq data confirms minimal translation from nauorf 2 (starting with AUA) of the GCN4 mrna, there are indications that other 5'-proximal elements in its 5' UTR might have a stronger effect on translation. Two additional sites of SSU accumulation (SSU clusters) are observed upstream of uorfs 1-4 with overall SSU occupancy comparable to the uorf 1 start codon, however, footprints in these regions are longer, typical of scanning SSU, and the corresponding SSU footprint counts do 13

14 RESEARCH SUPPLEMENTARY INFORMATION not increase in the SYO1-TAP strain, indicating that these footprints are not related to start codon recognition (Fig. 3b; Extended Data Fig. 8a). Most intriguingly, such clustering of SSU in the 5' UTRs unrelated to uorf translation can result in significant effects on the protein production from mrna. This is well exemplified by YNR016C mrna for which translation of a uorf starting with AUG at 342 nt from the main ORF start was identified by ribosome profiling 136. While some minimal SSU accumulation on this start site is indeed detected by TCP-seq, two far more prominent clusters of SSU are located downstream of it approximately between positions 250 to 200 and 150 to 100 nt (Extended Data Fig. 8b). These clusters contain longer footprints compared to the ones that derive from start codons and do not intensify in the SYO1-TAP strain (Extended Data Fig. 8b). Because the total footprint count in these clusters exceeds start codon footprint count for both WT and SYO1-TAP cells, these regions of YNR016C mrna probably limit its translational output and thus can be considered as candidate effectors of translational control. Other mrnas with similar uorf-unrelated impeded scanning and strong effect on translation efficiency can be identified from the TCP-seq dataset (Fig. 3c upper band; Supplementary Table 2; other mrnas in Extended Data Fig. 8). Interestingly, mrna of yeast poly(a) binding protein (PAB1), with a relatively short 5' UTR, demonstrates impediments of scanning and clustering of scanning SSU in the region from 75 to 45 nt from the ORF start (Fig. 3a; Extended Data Fig. 8b). This region of the PAB1 mrna immediately precedes an A- rich sequence located further downstream of it up to the start codon of the PAB1 ORF (Fig. 3a). Pab1 protein could bind to the A-rich sequence of its mrna resulting in a negative feedback regulation of its synthesis as proposed in the initial study characterising poly(a) binding properties of Pab Although there are no studies in yeast demonstrating this effect directly, an inhibitory role of Pab1 on mrnas with poly(a) stretches was inferred from genome-wide translation measurements. While the PAB1 mrna 5' UTR possesses 11 consecutive adenine nucleotides, one short of the 12 consecutive residues proposed to be required for the full inhibitory effect 150, it was demonstrated that Pab1 does still associate with its own mrna 149. Further, while the 5' UTR sequences of yeast PAB1 and mammalian PABP mrnas are not homologous overall, there still is an autoregulatory A-rich sequence (ARS) present in PABP mrna 151. Moreover, it was shown that under conditions of decreased capdependent translation initiation, PAB1 mrna undergoes cap-independent but eif4gdependent internal translation initiation that is thought to be a result of Pab1 binding to the A- rich sequences in yeast 18. These facts, combined with the TCP-seq data showing SSU accumulation on PAB1 mrna immediately upstream of its A-rich sequence, raise an interesting possibility of PAB1 mrna regulation where suppression of Pab1 production under normal conditions and relatively high translation of PAB1 mrna during stress results in condition-adjusted synthesis of Pab1. Overall, using TCP-seq we observe translation regulation during ribosomal scanning of mrna that can be either canonical translational control by uorfs described before, or uorfunrelated impeded scanning of mrna, visualised as accumulation of scanning SSU footprints in clusters over 5' UTRs. We do not find widespread dependency of the SSU footprint cluster formation on the apparent mrna structure as measured by the PARS score 17. Determinants of formation of such SSU clusters could be complex, including sequence of mrna, binding of trans-acting factors, SSU pausing over mrna, abortive initiation or internal ribosomal 14

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