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, 163 (5), 1267-1280

Coupling of mRNA Structure Rearrangement to Ribosome Movement During Bypassing of Non-coding Regions

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Coupling of mRNA Structure Rearrangement to Ribosome Movement During Bypassing of Non-coding Regions

Jin Chen et al. Cell.

Abstract

Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass a region of 50 nt, resuming translation 3' of this gap. How this large-scale, specific hop occurs and what determines whether a ribosome bypasses remain unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4's gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans mRNA in search of optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements.

Figures

Figure 1
Figure 1. Dynamic pathways of gene60 bypassing
(A) The elements of gene 60 bypass are labeled: (1) the UAG stop codon immediately 3’ to the take-off GGA site at codon Gly45, (2) the tRNAGly and the matching GGA take-off and landing sites, (3) an upstream nascent peptide signal, (4) a stem-loop consisting of the take-off codon, and possibly (5) a GAG Shine-Dalgarno-like sequence located 6 nucleotides 5’ to the landing site to promote precision of landing. Full sequence of the gene 60 mRNA is shown, where first 42 codons written as their amino acids (with Met being codon 0) and the remaining sequence labeled with nucleotides. The coloring of the codon or nucleotide matches the coloring in part B and C. (B) Representative traces of ribosomes Cy3B (green) fluorescent intensity for bypassed and non-bypassed ribosomes. For both cases, there is a phase with normal translation (labeled with a green line), a phase of slow down (blue line), and either terminating at a stop codon for non-bypassed ribosomes or go into a rotated state pause at codon Gly45 for bypassed ribosomes. The state assignment is shown in red, with the codon counts above. (C) The mean state lifetimes. The first 39 codons, when translation occurs normally, are colored in green. Codons 40 to 44, characterized by slow-down due to nascent peptide interaction, are shown in blue. The take-off site at codon 45 is colored in red. At codon 45, there is a long rotated state pause. Codons after bypass are shaded in pink. Number of molecules analyzed, n = 451. (D) We can parse the subpopulation of ribosomes into bypassed and non-bypassed and separate the lifetimes shown in (C) into the two populations, giving us a bypassing efficiency of 35%. Only the bypassed ribosomes exhibit an increase in rotated state lifetime at codon Gly45. The color scheme is the same as (C). n = 451. See also Figure S1, Figure S2, and Figure S3.
Figure 2
Figure 2. Mutation of the nascent peptide interaction abolishes the slow-down
(A) In vivo analysis of bypassing with mutants of the nascent peptide. The absolute value of bypassing in these assays by the WT (2nd from left) is 33% and all other values are of a percentage of it. (B) Deleting the key interaction of the nascent peptide signal (KKYK) to AAAA do not increase non-rotated and rotated state lifetimes. Most ribosomes terminate at the stop codon after codon Gly45. An example trace is shown. The color scheme is the same as Figure 1. n = 424. (C) In vivo analysis of bypassing with fusions of gene 60/SecM nascent peptides. The cassette used to generate the result in the middle lane has gene 60 sequence encoding amino acids 32 to 46 in its native location 5’ adjacent to the gene 60 take-off codon. The SecM nascent peptide signal encoding sequence is 5’ adjacent to it. The right lane derives from a cassette with the SecM nascent peptide encoding sequence 5’ adjacent to the gene 60 take-off codon. See also Figure S4.
Figure 3
Figure 3. The –UUCG– hairpin stem loop, especially the top base-pairs, is important for the rotated state pause
(A) The hairpin is shown in green, and the UUCG tetraloop is marked in red. To investigate the role of the mRNA hairpin, the base pairs were disrupted; the increase in non-rotated state lifetime due to the nascent peptide signal is still observed, but long rotated state pause at Gly45 characteristic of bypassing is no longer detected. n = 244. (B) Mutation of 3 base pairs below the UUCG tetraloop decreased bypass efficiency to 12%. n = 442. (C) Mutation of the bottom portion of the hairpin. The bypass efficiency remained the same at 36%. n = 349.
Figure 4
Figure 4. Effects of the 5’ stem-loop
Synonymous mutations (shown in red) of the 5’ stem-loop (wild-type sequence shown in blue) to destabilize the secondary structure. The bypass efficiency decreased to 11.8%, with a corresponding decrease in rotated state lifetime at codon Gly45, suggesting that the 5’ stem-loop is important. n = 488. See also Figure S5.
Figure 5
Figure 5. Timing and mechanism of take-off and landing
(A) Using the Asp44Phe and Leu46Phe mutant mRNAs introduced in Figure S2, the timing of bypass was probed. Using the Asp45Phe mutant, we can get the timing of when the Cy5-tRNAPhe (red) departs relative to the start of the rotated state pause at Gly45. This gives an upper estimate of when translocation occurs during the pause. The mean departure time is 28.1 ± 8.5 s, which is a lot shorter than the mean lifetime of the pause (90 s), indicating that the translocation is uncoupled with reverse-rotation. This gives an estimate of when the launch occurs. (B) With Leu46Phe mutant, A-site accessibility could be probed with Cy5-tRNAPhe, giving an estimate on when landing is completed. The mean arrival time is 67.3 ± 13.0 s, which is also during the pause. Thus, bypass and landing is completed during the rotated state pause, making the A site available for tRNA binding. (C) Landing site was changed from GGA(Gly) to GUA(Val), mRNA sequence is shown. The increase in non-rotated state lifetime due to the nascent peptide signal can be seen. The rotated state pause at Gly45 is shorter than for wild-type. This is due to loss of Cy3B signal during the rotated state pause, when the ribosome fails to find the correct landing codon after launching the bypass and drops-off. Thus, matching take-off and landing codons are required. Consistent with this the percentage of ribosomes undergoing the rotated state pause at Gly45 is the same as wild-type. However, the percentage of ribosomes that resume after the pause is much lower. n = 469. (D) The end-states of the ribosome after the pause can be parsed to (1) loss of Cy3B signal (due to ribosome drop-off or photobleaching), (2) resume of translation after the pause, (3) end of movie during the pause, (4) return of the Cy3B signal (photobleaching of FRET quencher or dissociation of 50S), and (5), reverse-rotates but translation does not resume. (E) Both the take-off and landing codons where changed from wild-type GGA(Gly) to GUA(Val). The non-rotated and rotated state lifetimes for the double mutant. Very similar behavior to the landing site mutant can be seen. Thus, the identity of the take-off codon is not critical for initiating bypass. However, for successful landing, the identity of the tRNA is very important. n = 466. See also Figure S6.
Figure 6
Figure 6. Timing of ribosomal bypassing and scanning monitored by ribosome-mRNA FRET
(A) For ribosome-mRNA FRET to monitor the hop, the 30S subunit was labeled with Cy3B on helix 33a, near the beak of 30S subunit, and the mRNA is labeled with Cy5 downstream of the landing site. Landing after bypassing brings the ribosome within FRET distance to the Cy5 dye. (B) Asp44Phe mutant mRNA allows us to use Cy3.5-labeled (yellow) to track when the tRNA departs at codon 44. This represents the timing of uncoupled translocation during the rotated state pause at Gly45. The ribosome bypasses on average 3.4 ± 0.9 s after uncoupled translocation. (C) Translation of the Leu46Phe mutant mRNA allows us to use Cy3.5-labeled (yellow) to track when the tRNA arrives to the A site after the bypass. This represents the timing of when successful landing occurs and the A site is available after the bypass (on average 50.5 ± 13.0 s). (D) With the use of the +15 Cy5-oligo (15 nucleotides downstream of the GGA landing codon, the same used for (B) and (C)), the FRET lifetime is 72.3 ± 20.0 s. By moving the Cy5-oligonucleotide to 3 nucleotides downstream of the take-off GGA codon (called +3 Cy5-oligo) such that the ribosome footprint is blocked upon landing, the FRET average lifetime decreases significantly to 10.2 ± 4.5 s. Since there is still a stable FRET signal, the ribosome must land upstream of the oligonucleotide, then scan to find the landing site, during which the ribosome contact quenches the Cy5 dye.
Figure 7
Figure 7. Model of translational bypassing
At the take-off Gly45 (GGA) codon, after the arrival of tRNAGly to the A site and peptidyl transfer, the ribosome rotates. The nascent peptide signal interaction pulls on the peptidyl-tRNA, as indicated by the red arrow. The 5’ stem-loop is shown in blue, the bypass hairpin is shown in green, and the UUCG tetraloop is shown in red. EF-G catalyzes translocation, moving the GGA codon to the P site. Combined with the propensity of the UUCG tetraloop to re-fold, the ribosome slips forward and leads to uncoupled translocation, allowing the UUCG tetraloop and a few base pairs to re-fold within the A site and the 5’ stem-loop to completely refold. Since the 5’ stem-loop blocks backwards movement, relaxation of the unstable state threads the mRNA in the 5’ direction. Refolding of the bypass hairpin launches ribosome forward. The ribosome then scans the mRNA to find the optimal base pairing, assisted by the GAG Shine-Dalgarno-like sequence and a possible 3’ stem-loop. Upon landing at the landing-site, the next tRNA accommodates to the rotated ribosome to help re-define the reading frame, resuming translation. See also Figure S7.

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