Translational frameshifting involves the repositioning of ribosomes on their messages into decoding frames that differ from those dictated during initiation. Some messenger RNAs (mRNAs) contain motifs that promote deliberate frameshifting to regulate production of the encoded proteins. The mechanisms of frameshifting have been investigated in many systems, and the resulting models generally involve single ribosomes responding to stimulator sequences in their engaged mRNAs. We discovered that the abundance of ribosomes on messages containing the
IS3, dnaX, and prfB frameshift motifs significantly influences the levels of frameshifting. We show that this phenomenon results from ribosome collisions that occur during translational stalling, which can alter frameshifting in both the stalled and trailing ribosomes. Bacteria missing ribosomal protein bL9 are known to exhibit a reduction in reading frame maintenance and to have a strong dependence on elongation factor P (EFP). We discovered that ribosomes lacking bL9 become compacted closer together during collisions and that the E-sites of the stalled ribosomes appear to become blocked, which suggests subsequent transpeptidation in transiently stalled ribosomes may become compromised in the absence of bL9. In addition, we determined that bL9 can suppress frameshifting of its host ribosome, likely by regulating E-site dynamics. These findings provide mechanistic insight into the behavior of colliding ribosomes during translation and suggest naturally occurring frameshift elements may be regulated by the abundance of ribosomes relative to an mRNA pool.
bL9; dnaX; frameshift; ribosome; translation.
Copyright © 2019 the Author(s). Published by PNAS.
Conflict of interest statement
The authors declare no competing interest.
Ribosome abundance on an mRNA influences frameshifting. A reporter construct based on the
IS3 pseudoknot was used to evaluate the impact of ribosome load on −1 frameshifting. ( A) Schematic of the reporter showing the location of the SD sequence that was altered to adjust translation initiation efficiency, the mOrange2 protein domain, out-of-frame ClpXP degrons (gray “deg”), a His tag, a test sequence (green bar), the 6 IS3 pseudoknot stimulator, and a FLAG tag in the −1 frame. Stop codons for the 0 and −1 reading frames are marked with red octagons. The test sequence is displayed below, with the encoded amino acids in the 0 (top) and −1 (bottom) frames and slippery tetrad nucleotides in red. ( B) Anti-His 6 Western blot revealing the products produced from a mock culture, a nonslippery reporter version lacking the slippery tetrad (A_AAG to C_AAG), a hard-coded “frameshift” product, and a slippery reporter with a strong consensus SD sequence. The canonical normal product and −1 frameshifted products are indicated. ( C) ( Left) Western blot of reporters with SD sequences altered to reduce translation initiation. As the translation initiation efficiency waned, the percentage of frameshifted product increased. ( Right) Bar graph of frameshifted product percentages from separate quantitative Westerns of 3 biological replicates. Error bars are 1 SD. Asterisks signify t test P values < 0.05 (*) and <0.001 (***). ( D) Sucrose gradients of lysates prepared from cells that had expressed the reporters with the strongest and weakest SD were used to separate ribosome forms prior to extracting RNA for reverse-transcription RT-qPCR. An absorbance profile is shown in gray with the locations of the 70S monosomes and polysome peaks. Primers that amplified the 5′ ends of the reporter mRNAs and 16S rRNA were used to establish the message/ribosome ratios, which were normalized to the amount of strong mRNA observed in the 70S peak. Error bars represent 1 SD of 3 experimental replicates.
Frameshifting at the
dnaX and prfB motifs. ( A) Schematics of the dnaX and prfB programmed frameshift motifs that were cloned into the reporter system. The upstream SD-like stimulators are shown in blue, with their deactivated versions below. The 0-frame amino acids are shown above and the frameshifted amino acids are shown below each sequence. Stop codons are red octagons. The dnaX motif also contains a downstream stem loop stimulator that was not altered. Note that, for the dnaX motif, −1 frameshifting produced the smaller translation product. ( B) Frameshifting at the dnaX motif as a function of ribosome load. The construct with a strong SD sequence (AGGAGG) exhibited more frameshifting than a version with a weak SD sequence (AGATGG). Inactivation of the nearby SD-like stimulator reduced frameshifting and increased the influence of ribosome load. ( Left) To even the abundances for presentation clarity, the anti-His 6 Western had the strong samples diluted 35- and 25-fold for the with stimulator and without stimulator samples, respectively. ( Right) Separate Westerns of 3 biological replicates were used to generate the data for the bar chart. ( C) Frameshifting at the prfB motif. The construct with a strong SD sequence (AGGAGG) exhibited less frameshifting than the version with a weak SD sequence (AGAAGG); ( Left) the strong versions were diluted 10-fold for the presented Western. Inactivation of the nearby SD-like stimulator reduced frameshifting and increased the influence of ribosome load. ( Right) Separate Western blots of 3 biological replicates were used to generate the data for the bar chart. Error bars represent 1 SD. Asterisks indicate t test P values of <0.01 (**), <0.001 (***), and <0.0001 (****).
Ribosome load alters frameshifting in a cell-free translation system. An mRNA was designed to test the effect of ribosome load on frameshifting in a purified translation system. (
A) Schematic of the reporter mRNA showing the IS3 element (detailed in Fig. 1) located between the coding regions for Firefly luciferase and an out-of-frame NanoLuc luciferase. ( B) Reporter mRNA that was synthesized in vitro using T7 RNA polymerase was added at different final concentrations to translation reactions containing purified components (∼2 μM ribosomes). After 2 h of translation, the luminescence of each luciferase was independently measured in a 96-well plate. The plate was imaged, the 10-min-averaged luminescence values were subsequently recorded in a luminometer, and those values from experimental replicates were then averaged. Representative plots of luminescence signals vs. time are shown below wells of a plate after subtracting background signals from the no-RNA controls to illustrate signal stabilities. The NanoLuc signal was substantially brighter than the Firefly signal. ( C) Plot of averaged data from 3 experimental replicates. The NanoLuc (Nluc)/Firefly ratio increased significantly as the mRNA abundance increased, which reduced ribosome load per message. Asterisks indicate a t test P value of <0.01 (**).
Stalled ribosomes are frameshift stimulators. A series of reporters was constructed to evaluate the influence of ribosome collisions on −1 frameshifting. (
A, Left) Schematic of a collision between a ribosome transiently stalled at a stop codon (red octagon) and a trailing ribosome engaged with a slippery sequence located at different upstream positions (green). ( Right) A detailed schematic. A slippery heptad sequence was positioned at varying distances 5′ to a slowly decoded UGA stop codon (red octagon). Nonslippery versions contained changes (blue) that hampered repositioning of the tRNAs. A downstream FLAG-tag appendage was encoded in the −1 frame. Out-of-frame ClpXP degrons preceded the test regions. The distance between the slippery sequence and the stalling stop codon was varied by inserting additional Ser codons such that the final A-site to A-site spacing ranged from 5 to 11 codons. ( B) Anti-His 6 Western blots of the reporter series that was expressed in bL9+ and bL9− cells. Frameshifting was evident in each case, but peaked with a 9-codon spacer in bL9+ and with an 8-codon spacer in bL9− cells. ( C) Quantifications from 2 biological replicates were averaged and plotted. The peak frameshifting levels were significantly higher than the levels with the 5-codon spacer, and the bL9− cells exhibited more frameshifting overall. P values < 0.01(**) and <0.001 (***) are indicated. ( D) ( Top) Anti-FLAG and ( Bottom) anti-His 6 Westerns showing comparisons between reporters with 5 or 9 spacer codons with and without the slippery heptad.
Ribosomal protein bL9 alters interribosomal spacing. A construct was designed to stably stall ribosomes so that the positions of trailing ribosomes could be established by characterizing their nascent peptides. (
A) Schematic of 2 colliding ribosomes and the nascent peptide sequences. The lead ribosome is stably engaged with a SecM stalling motif with peptide-tRNA Gly in the P-site and tRNA Pro in the A-site (red). The colliding ribosome contains a shorter nascent peptide, with deacylated tRNA Ile in the P/E-site and peptide-tRNA Ser in the A/P-site (blue). The peptides contained N-terminal His 6 tags that allowed for purification under denaturing conditions prior to mass spectroscopy. ( B) MALDI-TOF mass spectra of peptides recovered from sucrose gradient polysome fractions of bL9+ (blue) and bL9− cells (red, inverted). The dominant mass for the SecM-stalled polysomes corresponded to a peptide ending at the Gly residue of the SecM sequence shown in A (theoretical m/z = 4,801.40 Da, observed = 4,801.14 Da). Peptides recovered from bL9+ polysomes also contained a prominent mass corresponding to a peptide that was 8 residues shorter than the SecM-stalled peptide (theoretical = 4,019.53 Da, observed = 4,019.42). The peptides from bL9− polysomes contained an additional peptide 7 residues shorter (theoretical = 4,147.66 Da, observed = 4,146.90 Da).
Stalled ribosomes have higher E-site occupancy in the absence of bL9. The SecM stalling construct was used to generate compacted polysomes that were recovered to quantify tRNA abundances in compacted polysomes. (
A) Schematic of a SecM collision complex with an E-site deacylated tRNA Ala1B (pink), a P-site tRNA Gly3 connected to the nascent peptide (celeste), and an A-site tRNA Pro2/3 waiting to engage the peptidyltransferase center (chartreuse). In the absence of bL9, trailing ribosomes advance one codon closer to stalled ribosomes (dashed outline) and potentially influence the egress of E-site tRNA Ala1B (red arrow). ( B) A plasmid encoding a SecM-stall peptide (41 residues including the stalling Gly) was expressed for 20 min in bL9+ and bL9− cells, and lysates were resolved on sucrose gradients and fractionated. The absorbance profiles of representative gradients are shown with the bL9+ material (blue) and bL9− material (red) normalized to the abundance of monosomes. The positions of the monosomes and compacted 4-somes are indicated. An overcompacted 5-some peak was reproducibly more abundant in bL9− lysates. ( C) RNA was purified from 4-some gradient peaks, converted to cDNA with dedicated primers, normalized, and used as templates in RT-qPCR reactions specific for the SecM-stall mRNA, as well as the tRNA Ala1B and tRNA Gly3 isoforms that had engaged the stalled ribosomes’ E- and P-site codons. For comparison, the level of SecM-stall mRNA and the ratio of tRNA Ala1B to tRNA Gly3 was normalized to the average amount found in the fractions recovered from bL9+ cells. Error bars represent SDs from 3 biological replicates and 5 qPCR measurements for each target RNA. A P value < 0.01 is indicated (**).
Ribosomal protein bL9 functions in
cis to reduce frameshifting. A frameshift reporter was designed to evaluate the performance of single ribosomes as they engage a slippery sequence. ( A) Schematic of reporters with a slippery heptad (red) located at the 5′ end of a mini ORF with a downstream NanoLuc ORF in the −1 frame. Transcripts containing either 27 or 5 nucleotides 5′ to the SD were compared. ( B) After expression in bL9+ and bL9− cells, NanoLuc luminescence was measured and normalized to the level present in bL9+ cells. The level of frameshifting was significantly higher in cells lacking bL9. Error bars are SDs of 3 biological replicates. P values < 0.0001 (****) are indicated.
Models of mRNA tension and collisions. (
A) Schematic of a ribosome in 2 locations relative to a frameshift element containing flanking stimulators. ( Left) The SD sequence in the mRNA (green) interacts with the anti-SD (orange) in a relaxed manner, and the downstream mRNA stem loop is relaxed. ( Right) A ribosome that has translocated farther into the motif, with a tensioned SD:anti-SD structure and a partially unfolded stem loop. The combination of the 2 flanking forces provides the energy to increase −1 frameshifting. A potential trailing ribosome is depicted as a dashed line. The trailing ribosome may promote entry of the lead ribosome into a compromised location while still allowing for a −1 frameshift. ( B) Ribosome collision models with a stalled ribosome and a trailing ribosome. ( Left) A cryo-EM structure of a ribosome awaiting translation termination (stalled, PDB ID 6ore; slate and orange) that is docked to a structure of a ribosome in the resting state (trailing, PDB 6osq; gold and light green) using a collision orientation similar to that observed in many crystal structures. Protein bL9 of the stalled ribosome is colored red, and protein uS4 of the trailing ribosome is colored pink. ( Center) Protein bL9 was removed while maintaining the orientations of both particles. ( Right) The trailing ribosome was reoriented and moved closer such that uS4 of the trailing ribosome accommodated the space previously occupied by bL9. This relocation reduced the intervening mRNA length by approximately one codon.
All figures (8)
Dynamic pathways of -1 translational frameshifting.
Nature. 2014 Aug 21;512(7514):328-32. doi: 10.1038/nature13428. Epub 2014 Jun 11.
24919156 Free PMC article.
Mechanism of tRNA-mediated +1 ribosomal frameshifting.
Proc Natl Acad Sci U S A. 2018 Oct 30;115(44):11226-11231. doi: 10.1073/pnas.1809319115. Epub 2018 Sep 27.
Proc Natl Acad Sci U S A. 2018.
30262649 Free PMC article.
Ribosomal frameshifting, jumping and readthrough.
Curr Opin Cell Biol. 1991 Dec;3(6):1051-5. doi: 10.1016/0955-0674(91)90128-l.
Curr Opin Cell Biol. 1991.
1814364 Free PMC article.
Changed in translation: mRNA recoding by -1 programmed ribosomal frameshifting.
Trends Biochem Sci. 2015 May;40(5):265-74. doi: 10.1016/j.tibs.2015.03.006. Epub 2015 Apr 4.
Trends Biochem Sci. 2015.
25850333 Free PMC article.
Polysomes Bypass a 50 Nucleotide Coding Gap less Efficiently than Monosomes Due to Attenuation of a 5' mRNA Stem Loop and Enhanced Drop-off.
J Mol Biol. 2020 May 23:S0022-2836(20)30346-6. doi: 10.1016/j.jmb.2020.05.010. Online ahead of print.
J Mol Biol. 2020.
32454154 Free PMC article.
Ribosome Dimerization Protects the Small Subunit.
J Bacteriol. 2020 Apr 27;202(10):e00009-20. doi: 10.1128/JB.00009-20. Print 2020 Apr 27.
J Bacteriol. 2020.
Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts.
EMBO J. 2020 Feb 3;39(3):e103365. doi: 10.15252/embj.2019103365. Epub 2019 Dec 20.
EMBO J. 2020.
Atkins J. F., Loughran G., Bhatt P. R., Firth A. E., Baranov P. V., Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use. Nucleic Acids Res. 44, 7007–7078 (2016).
Rozov A., Demeshkina N., Westhof E., Yusupov M., Yusupova G., New structural insights into translational miscoding. Trends Biochem. Sci. 41, 798–814 (2016).
Rodnina M. V., Translation in prokaryotes. Cold Spring Harb. Perspect. Biol. 10, a032664 (2018).
Chang B., Halgamuge S., Tang S. L., Analysis of SD sequences in completed microbial genomes: Non-SD-led genes are as common as SD-led genes. Gene 373, 90–99 (2006).
Studer S. M., Joseph S., Unfolding of mRNA secondary structure by the bacterial translation initiation complex. Mol. Cell 22, 105–115 (2006).
Research Support, N.I.H., Extramural
Escherichia coli / genetics*
Escherichia coli / metabolism
Frameshift Mutation / genetics
Frameshifting, Ribosomal / genetics*
Nucleic Acid Conformation
Peptide Elongation Factors / metabolism
Protein Biosynthesis / genetics
RNA, Messenger / genetics*
Reading Frames / genetics*
Ribosomal Proteins / metabolism
Peptide Elongation Factors