Ribosomal pause

{{Short description|Queueing of ribosomes during protein synthesis}} [[File:Peptide syn.svg|thumb|The ribosome assembles polymeric protein molecules whose sequence is controlled by the sequence of messenger RNA molecules. This is required by all living cells and associated viruses.]] '''Ribosomal pause''' refers to the queueing or stacking of ribosomes during translation of the nucleotide sequence of mRNA transcripts. These transcripts are decoded and converted into an amino acid sequence during protein synthesis by ribosomes. Due to the pause sites of some mRNAs, there is a disturbance caused in translation.<ref name="Gawroński 2557–2569">{{cite journal | vauthors = Gawroński P, Jensen PE, Karpiński S, Leister D, Scharff LB | title = Pausing of Chloroplast Ribosomes Is Induced by Multiple Features and Is Linked to the Assembly of Photosynthetic Complexes | journal = Plant Physiology | volume = 176 | issue = 3 | pages = 2557–2569 | date = March 2018 | pmid = 29298822 | pmc = 5841727 | doi = 10.1104/pp.17.01564 }}</ref> Ribosomal pausing occurs in both eukaryotes and prokaryotes.<ref name="Li 2012 538–541">{{cite journal | vauthors = Li GW, Oh E, Weissman JS | title = The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria | journal = Nature | volume = 484 | issue = 7395 | pages = 538–41 | date = March 2012 | pmid = 22456704 | pmc = 3338875 | doi = 10.1038/nature10965 | bibcode = 2012Natur.484..538L | url = https://biobabel.wordpress.com/2012/04/27/on-ribosomal-pausing/ }}</ref><ref name="pmid10648594">{{cite journal | vauthors = Lopinski JD, Dinman JD, Bruenn JA | title = Kinetics of ribosomal pausing during programmed -1 translational frameshifting | journal = Molecular and Cellular Biology | volume = 20 | issue = 4 | pages = 1095–103 | date = February 2000 | pmid = 10648594 | pmc = 85227 | doi = 10.1128/MCB.20.4.1095-1103.2000 }}</ref> A more severe pause is known as a '''ribosomal stall'''.<ref name="pmid28138069">{{cite journal |last1=Buskirk |first1=Allen R. |last2=Green |first2=Rachel |title=Ribosome pausing, arrest and rescue in bacteria and eukaryotes |journal=Philosophical Transactions of the Royal Society B: Biological Sciences |date=19 March 2017 |volume=372 |issue=1716 |article-number=20160183 |doi=10.1098/rstb.2016.0183 |pmid=28138069 |pmc=5311927}}</ref>

It's been known since the 1980s that different mRNAs are translated at different rates. The main reason for these differences was thought to be the concentration of varieties of rare tRNAs limiting the rate at which some transcripts could be decoded.<ref name="pmid11713298">{{cite journal | vauthors = Kontos H, Napthine S, Brierley I | title = Ribosomal pausing at a frameshifter RNA pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency | journal = Molecular and Cellular Biology | volume = 21 | issue = 24 | pages = 8657–70 | date = December 2001 | pmid = 11713298 | pmc = 100026 | doi = 10.1128/MCB.21.24.8657-8670.2001 }}</ref> However, with research techniques such as ribosome profiling, it was found that at certain sites there were higher concentrations of ribosomes than average, and these pause sites were tested with specific codons. No link was found between the occupancy of specific codons and amount of their tRNAs. Thus, the early findings about rare tRNAs causing pause sites don't seem plausible.<ref name="Li 2012 538–541"/>

Two techniques can localize the ribosomal pause site in ''vivo'''';'' a micrococcal nuclease protection assay and isolation of polysomal transcript.<ref name="pmid22806127">{{cite journal | vauthors = Jha SS, Komar AA | title = Isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mRNA | journal = Journal of Visualized Experiments | issue = 65 | date = July 2012 | pmid = 22806127 | pmc = 3471273 | doi = 10.3791/4026 }}</ref> Isolation of polysomal transcripts occurs by centrifuging tissue extracts through a sucrose cushion with translation elongation inhibitors, for example cycloheximide.<ref>{{cite journal | vauthors = Kim JK, Hollingsworth MJ | title = Localization of in vivo ribosome pause sites | journal = Analytical Biochemistry | volume = 206 | issue = 1 | pages = 183–8 | date = October 1992 | pmid = 1456432 | doi = 10.1016/s0003-2697(05)80031-4 }}</ref>

Ribosome pausing can be detected during preprolactin synthesis on free polysomes, when the ribosome is paused the other ribosomes are tightly stacked together. When the ribosome pauses, during translation, the fragments that started to translate before the pause took place are overrepresented. However, along with the mRNA if the ribosome pauses then specific bands will be improved in the trailing edge of the ribosome.<ref name="Ribosome pausing and stacking durin">{{cite journal | vauthors = Wolin SL, Walter P | title = Ribosome pausing and stacking during translation of a eukaryotic mRNA | journal = The EMBO Journal | volume = 7 | issue = 11 | pages = 3559–69 | date = November 1988 | pmid = 2850168 | pmc = 454858 | doi = 10.1002/j.1460-2075.1988.tb03233.x }}</ref>

Some of the elongation inhibitors, such as: cycloheximide (in eukaryotes) or chloramphenicol, cause the ribosomes to pause and to accumulate in the start codons. Elongation Factor P regulates the ribosomal pause at polyproline in bacteria, and when there is no EFP the density of ribosomes decreases from the polyproline motifs. If there are multiple ribosome pauses, then the EFP won't resolve it.<ref name="Brar 552–557">{{cite journal | vauthors = Brar GA, Yassour M, Friedman N, Regev A, Ingolia NT, Weissman JS | title = High-resolution view of the yeast meiotic program revealed by ribosome profiling | journal = Science | volume = 335 | issue = 6068 | pages = 552–7 | date = February 2012 | pmid = 22194413 | pmc = 3414261 | doi = 10.1126/science.1215110 | bibcode = 2012Sci...335..552B }}</ref>

== Resolution and effects on gene expression == Some forms of ribosomal pause are reversible without needing to discard the translated peptide and mRNA. This sort, usually described as a slowdown, is usually caused by polyproline stretches (resolved by EFP or eIF5A) and uncharged tRNA.<ref name="pmid28138069"/> Slowdowns are important for the cell to control how much protein is produced;<ref name=":13">{{cite journal | vauthors = Darnell AM, Subramaniam AR, O'Shea EK | title = Translational Control through Differential Ribosome Pausing during Amino Acid Limitation in Mammalian Cells | language = en | journal = Molecular Cell | volume = 71 | issue = 2 | pages = 229–243.e11 | date = July 2018 | pmid = 30029003 | doi = 10.1016/j.molcel.2018.06.041 | pmc = 6516488 }}</ref> it also aids co-translational folding of the nascent polypeptide on the ribosome, and delays protein translation while its encoding mRNA; this can trigger ribosomal frameshifting.<ref name="pmid17696878" />

More severe "stalls" can be caused an actual lack of tRNA or by the mRNA terminating without a stop codon.<ref name="pmid28138069"/> In this case, ribosomal quality control (RQC) performs ''crisis rescue'' by translational abandonment. This releases the ribosome from the mRNA. The incomplete polypeptide is targeted for destruction; in eukaryotes, mRNA no-go decay is also triggered.<ref name="pmid17696878">{{cite journal | vauthors = Buchan JR, Stansfield I | title = Halting a cellular production line: responses to ribosomal pausing during translation | journal = Biology of the Cell | volume = 99 | issue = 9 | pages = 475–87 | date = September 2007 | pmid = 17696878 | doi = 10.1042/BC20070037 | doi-access = free }}</ref>

It is difficult for RQC machinery to differentiate between a slowdown and a stall. It is possible for a mRNA sequence that normally produces a protein slowly to produce nothing instead due to intervention by RQC under different conditions.<ref>{{cite journal | vauthors = Collart MA, Weiss B | title = Ribosome pausing, a dangerous necessity for co-translational events | journal = Nucleic Acids Research | volume = 48 | issue = 3 | pages = 1043–1055 | date = February 2020 | pmid = 31598688 | doi = 10.1093/nar/gkz763 | pmc = 7026645 }}</ref>

=== Rescue mechanisms === In bacteria, three rescue mechanisms are known. * The main, universal system involves transfer-messenger RNA (tmRNA) and SmpB. The tRNA first binds to the ribosome like a tRNA, then with SmpB's help shifts into the mRNA position to translate a short peptide ending on a normal stop codon.<ref name="pmid28138069"/> * Alternative ribosome-rescue factor A (ArfA) is an alternative system in ''E. coli''. It recruits RF2.<ref name="pmid28138069"/> * Alternative ribosome-rescue factor B (ArfB) is another alternative from ''E. coli''. It works like a GGQ-release factor itself, releasing the peptide from tRNA.<ref>{{cite journal |last1=Chan |first1=KH |last2=Petrychenko |first2=V |last3=Mueller |first3=C |last4=Maracci |first4=C |last5=Holtkamp |first5=W |last6=Wilson |first6=DN |last7=Fischer |first7=N |last8=Rodnina |first8=MV |title=Mechanism of ribosome rescue by alternative ribosome-rescue factor B. |journal=Nature Communications |date=14 August 2020 |volume=11 |issue=1 |page=4106 |doi=10.1038/s41467-020-17853-7 |pmid=32796827|pmc=7427801 |bibcode=2020NatCo..11.4106C }}</ref> At the same time, it fits into the mRNA tunnel to remove the mRNA.<ref>{{cite journal |last1=Carbone |first1=Christine E. |last2=Demo |first2=Gabriel |last3=Madireddy |first3=Rohini |last4=Svidritskiy |first4=Egor |last5=Korostelev |first5=Andrei A. |title=ArfB can displace mRNA to rescue stalled ribosomes |journal=Nature Communications |date=December 2020 |volume=11 |issue=1 |page=5552 |doi=10.1038/s41467-020-19370-z|pmid=33144582 |pmc=7641280 |bibcode=2020NatCo..11.5552C }}</ref>

In eukaryotes, the main mechanism involves PELO:HBS1L.<ref name="pmid28138069"/>

== Advantage of the ribosomal pause == thumb|Structure of the Ribosome When the ribosome movement on the mRNA is not linear, the ribosome gets paused at different regions without a precise reason. The ribosome pause position will help to identify the mRNA sequence features, structure, and the transacting factor that modulates this process.<ref name="pmid2850168">{{cite journal | vauthors = Wolin SL, Walter P | title = Ribosome pausing and stacking during translation of a eukaryotic mRNA | journal = The EMBO Journal | volume = 7 | issue = 11 | pages = 3559–69 | date = November 1988 | pmid = 2850168 | pmc = 454858 | doi = 10.1002/j.1460-2075.1988.tb03233.x}}</ref> The advantage of ribosomal pause sites that are located at protein domain boundaries are aiding the folding of a protein.<ref name="Gawroński 2557–2569" /> There are times when the ribosomal pause does not cause an advantage and it needs to be restricted. In translation, elF5A inhibits ribosomal pausing for translation to function better. Ribosomal pausing can cause more non-canonical start codons without elF5A in eukaryotic cells. When there is a lack of elF5A in the eukaryotic cell, it can cause an increase in ribosomal pausing.<ref>{{cite journal | vauthors = Manjunath H, Zhang H, Rehfeld F, Han J, Chang TC, Mendell JT | title = Suppression of Ribosomal Pausing by eIF5A Is Necessary to Maintain the Fidelity of Start Codon Selection | journal = Cell Reports | volume = 29 | issue = 10 | pages = 3134–3146.e6 | date = December 2019 | pmid = 31801078 | pmc = 6917043 | doi = 10.1016/j.celrep.2019.10.129 }}</ref> The ribosomal pausing process can also be used by amino acids to control translation.<ref name=":13"/>

== The location of the ribosome pause event in ''vitro'' == It is known that ribosomes pause at distinct sites, but the reasons for these pauses are mostly unknown. Also, the ribosome pauses if the pseudoknot is disrupted. 10% of the ribosome pauses at the pseudoknot and 4% of the ribosomes are terminated. Before the ribosome is obstructed it passes the pseudoknot.<ref>{{cite journal | vauthors = Somogyi P, Jenner AJ, Brierley I, Inglis SC | title = Ribosomal pausing during translation of an RNA pseudoknot | journal = Molecular and Cellular Biology | volume = 13 | issue = 11 | pages = 6931–40 | date = November 1993 | pmid = 8413285 | pmc = 364755 | doi = 10.1128/mcb.13.11.6931 }}</ref> An assay was put together by a group from the University of California in an effort to show a model of mRNA. The translation was monitored in two in vitro systems. It was found that translating ribosomes aren't uniformly distributed along an mRNA.<ref name="Ribosome pausing and stacking durin"/> Protein folding ''in vivo'' is also important and is related to protein synthesis. For finding the location of the ribosomal pause ''in vivo'', the methods that have been used to find the ribosomal pause ''in vitro'' can be changed to find these specific locations ''in vivo.''<ref name="pmid22806127" />

== Ribosome profiling == Ribosome profiling is a method that can reveal pausing sites through sequencing the ribosome protected fragments (RPFs or footprints) to map ribosome occupancy on the mRNA. Ribosome profiling has the ability to reveal the ribosome pause sites in the whole transcriptome. When the kinetics layer is added,<ref>{{cite journal|doi=10.1128/mcb.20.4.1095-1103.2000|title=Kinetics of Ribosomal Pausing during Programmed −1 Translational Frameshifting|year=2000|last1=Lopinski|first1=John D.|last2=Dinman|first2=Jonathan D.|last3=Bruenn|first3=Jeremy A.|journal=Molecular and Cellular Biology|volume=20|issue=4|pages=1095–1103|pmid=10648594|pmc=85227}}</ref> it discloses the time of the pause, and the translation takes place.<ref name="Brar 552–557"/> Ribosome profiling is however still in early stages and has biases that need to be explored further.<ref name="pmid28138069"/> Ribosome profiling allows for translation to be measured more accurately and precisely. During this process, translation needs to be stopped in order for ribosome profiling to be performed. This may cause a problem with ribosome profiling because the methods that are used to stop translation in an experiment can impact the outcome, which causes incorrect results. Ribosome profiling is useful for getting specific information on translation and the process of protein synthesis.<ref name=":0">{{cite journal | vauthors = Brar GA, Weissman JS | title = Ribosome profiling reveals the what, when, where and how of protein synthesis | journal = Nature Reviews. Molecular Cell Biology | volume = 16 | issue = 11 | pages = 651–64 | date = November 2015 | pmid = 26465719 | pmc = 5522010 | doi = 10.1038/nrm4069 }}</ref>

== References == {{reflist}}

== External links == * [http://www.ekevanbatenburg.nl/PKBASE/ Pseudobase] * [http://recode.ucc.ie Recode]

Category:RNA Category:Gene expression Category:Cis-regulatory RNA elements Category:Molecular genetics