Ribosomal pause

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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. Peptide syn.svg
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 mRNA's, there is a disturbance caused in translation. [1] Ribosomal pausing occurs in both eukaryotes and prokaryotes. [2] [3] A more severe pause is known as a ribosomal stall. [4]

Contents

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. [5] 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. [2]

Two techniques can localize the ribosomal pause site in vivo ; a micrococcal nuclease protection assay and isolation of polysomal transcript. [6] Isolation of polysomal transcripts occurs by centrifuging tissue extracts through a sucrose cushion with translation elongation inhibitors, for example cycloheximide. [7]

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. [8]

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. [9]

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. [4] Slowdowns are important for the cell to control how much protein is produced; [10] 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. [11]

More severe "stalls" can be caused an actual lack of tRNA or by the mRNA terminating without a stop codon. [4] 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. [11]

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. [12]

Rescue mechanisms

In bacteria, three rescue mechanisms are known.

In eukaryotes, the main mechanism involves PELO:HBS1L. [4]

Advantage of the ribosomal pause

Structure of the Ribosome Ribosome structure including subunits and binding sites.png
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. [15] The advantage of ribosomal pause sites that are located at protein domain boundaries are aiding the folding of a protein. [1] 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. [16] The ribosomal pausing process can also be used by amino acids to control translation. [10]

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. [17] 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. [8] 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. [6]

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, [18] it discloses the time of the pause, and the translation takes place. [9] Ribosome profiling is however still in early stages and has biases that need to be explored further. [19] 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. [20]

See also

Related Research Articles

<span class="mw-page-title-main">Ribosome</span> Synthesizes proteins in cells

Ribosomes are macromolecular machines, found within all cells, that perform biological protein synthesis. Ribosomes link amino acids together in the order specified by the codons of messenger RNA molecules to form polypeptide chains. Ribosomes consist of two major components: the small and large ribosomal subunits. Each subunit consists of one or more ribosomal RNA molecules and many ribosomal proteins. The ribosomes and associated molecules are also known as the translational apparatus.

<span class="mw-page-title-main">Translation (biology)</span> Cellular process of protein synthesis

In biology, translation is the process in living cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. This sequence is determined by the sequence of nucleotides in the RNA. The nucleotides are considered three at a time. Each such triple results in addition of one specific amino acid to the protein being generated. The matching from nucleotide triple to amino acid is called the genetic code. The translation is performed by a large complex of functional RNA and proteins called ribosomes. The entire process is called gene expression.

The 5′ untranslated region is the region of a messenger RNA (mRNA) that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes. While called untranslated, the 5′ UTR or a portion of it is sometimes translated into a protein product. This product can then regulate the translation of the main coding sequence of the mRNA. In many organisms, however, the 5′ UTR is completely untranslated, instead forming a complex secondary structure to regulate translation.

The Shine–Dalgarno (SD) sequence is a ribosomal binding site in bacterial and archaeal messenger RNA, generally located around 8 bases upstream of the start codon AUG. The RNA sequence helps recruit the ribosome to the messenger RNA (mRNA) to initiate protein synthesis by aligning the ribosome with the start codon. Once recruited, tRNA may add amino acids in sequence as dictated by the codons, moving downstream from the translational start site.

Ribosome shunting is a mechanism of translation initiation in which ribosomes bypass, or "shunt over", parts of the 5' untranslated region to reach the start codon. However, a benefit of ribosomal shunting is that it can translate backwards allowing more information to be stored than usual in an mRNA molecule. Some viral RNAs have been shown to use ribosome shunting as a more efficient form of translation during certain stages of viral life cycle or when translation initiation factors are scarce. Some viruses known to use this mechanism include adenovirus, Sendai virus, human papillomavirus, duck hepatitis B pararetrovirus, rice tungro bacilliform viruses, and cauliflower mosaic virus. In these viruses the ribosome is directly translocated from the upstream initiation complex to the start codon (AUG) without the need to unwind RNA secondary structures.

Bacterial translation is the process by which messenger RNA is translated into proteins in bacteria.

Eukaryotic translation is the biological process by which messenger RNA is translated into proteins in eukaryotes. It consists of four phases: initiation, elongation, termination, and recapping.

The Kozak consensus sequence is a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts. Regarded as the optimum sequence for initiating translation in eukaryotes, the sequence is an integral aspect of protein regulation and overall cellular health as well as having implications in human disease. It ensures that a protein is correctly translated from the genetic message, mediating ribosome assembly and translation initiation. A wrong start site can result in non-functional proteins. As it has become more studied, expansions of the nucleotide sequence, bases of importance, and notable exceptions have arisen. The sequence was named after the scientist who discovered it, Marilyn Kozak. Kozak discovered the sequence through a detailed analysis of DNA genomic sequences.

Eukaryotic initiation factors (eIFs) are proteins or protein complexes involved in the initiation phase of eukaryotic translation. These proteins help stabilize the formation of ribosomal preinitiation complexes around the start codon and are an important input for post-transcription gene regulation. Several initiation factors form a complex with the small 40S ribosomal subunit and Met-tRNAiMet called the 43S preinitiation complex. Additional factors of the eIF4F complex recruit the 43S PIC to the five-prime cap structure of the mRNA, from which the 43S particle scans 5'-->3' along the mRNA to reach an AUG start codon. Recognition of the start codon by the Met-tRNAiMet promotes gated phosphate and eIF1 release to form the 48S preinitiation complex, followed by large 60S ribosomal subunit recruitment to form the 80S ribosome. There exist many more eukaryotic initiation factors than prokaryotic initiation factors, reflecting the greater biological complexity of eukaryotic translation. There are at least twelve eukaryotic initiation factors, composed of many more polypeptides, and these are described below.

<span class="mw-page-title-main">Eukaryotic translation termination factor 1</span> Protein-coding gene in the species Homo sapiens

Eukaryotic translation termination factor1 (eRF1), also referred to as TB3-1 or SUP45L1, is a protein that is encoded by the ERF1 gene. In Eukaryotes, eRF1 is an essential protein involved in stop codon recognition in translation, termination of translation, and nonsense mediated mRNA decay via the SURF complex.

<span class="mw-page-title-main">Coronavirus frameshifting stimulation element</span>

In molecular biology, the coronavirus frameshifting stimulation element is a conserved stem-loop of RNA found in coronaviruses that can promote ribosomal frameshifting. Such RNA molecules interact with a downstream region to form a pseudoknot structure; the region varies according to the virus but pseudoknot formation is known to stimulate frameshifting. In the classical situation, a sequence 32 nucleotides downstream of the stem is complementary to part of the loop. In other coronaviruses, however, another stem-loop structure around 150 nucleotides downstream can interact with members of this family to form kissing stem-loops and stimulate frameshifting.

<span class="mw-page-title-main">HIV ribosomal frameshift signal</span>

HIV ribosomal frameshift signal is a ribosomal frameshift (PRF) that human immunodeficiency virus (HIV) uses to translate several different proteins from the same sequence.

A ribosome binding site, or ribosomal binding site (RBS), is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of translation. Mostly, RBS refers to bacterial sequences, although internal ribosome entry sites (IRES) have been described in mRNAs of eukaryotic cells or viruses that infect eukaryotes. Ribosome recruitment in eukaryotes is generally mediated by the 5' cap present on eukaryotic mRNAs.

Ribosomal frameshifting, also known as translational frameshifting or translational recoding, is a biological phenomenon that occurs during translation that results in the production of multiple, unique proteins from a single mRNA. The process can be programmed by the nucleotide sequence of the mRNA and is sometimes affected by the secondary, 3-dimensional mRNA structure. It has been described mainly in viruses, retrotransposons and bacterial insertion elements, and also in some cellular genes.

<span class="mw-page-title-main">Eukaryotic ribosome</span> Large and complex molecular machine

Ribosomes are a large and complex molecular machine that catalyzes the synthesis of proteins, referred to as translation. The ribosome selects aminoacylated transfer RNAs (tRNAs) based on the sequence of a protein-encoding messenger RNA (mRNA) and covalently links the amino acids into a polypeptide chain. Ribosomes from all organisms share a highly conserved catalytic center. However, the ribosomes of eukaryotes are much larger than prokaryotic ribosomes and subject to more complex regulation and biogenesis pathways. Eukaryotic ribosomes are also known as 80S ribosomes, referring to their sedimentation coefficients in Svedberg units, because they sediment faster than the prokaryotic (70S) ribosomes. Eukaryotic ribosomes have two unequal subunits, designated small subunit (40S) and large subunit (60S) according to their sedimentation coefficients. Both subunits contain dozens of ribosomal proteins arranged on a scaffold composed of ribosomal RNA (rRNA). The small subunit monitors the complementarity between tRNA anticodon and mRNA, while the large subunit catalyzes peptide bond formation.

<span class="mw-page-title-main">ALIL pseudoknot</span>

ALIL pseudoknot is an RNA element that induces frameshifting in bacteria. The expression of a minority of genes requires frameshifting to occur where the frequency of frameshifting is increased by a RNA secondary structure located on the 3' side of the shift site. This structure can be either a pseudoknot or a stem-loop and acts as a physical barrier to mRNA translocation so therefore causes ribosome pausing.

Translational regulation refers to the control of the levels of protein synthesized from its mRNA. This regulation is vastly important to the cellular response to stressors, growth cues, and differentiation. In comparison to transcriptional regulation, it results in much more immediate cellular adjustment through direct regulation of protein concentration. The corresponding mechanisms are primarily targeted on the control of ribosome recruitment on the initiation codon, but can also involve modulation of peptide elongation, termination of protein synthesis, or ribosome biogenesis. While these general concepts are widely conserved, some of the finer details in this sort of regulation have been proven to differ between prokaryotic and eukaryotic organisms.

Ribosome profiling, or Ribo-Seq, is an adaptation of a technique developed by Joan Steitz and Marilyn Kozak almost 50 years ago that Nicholas Ingolia and Jonathan Weissman adapted to work with next generation sequencing that uses specialized messenger RNA (mRNA) sequencing to determine which mRNAs are being actively translated. A related technique that can also be used to determine which mRNAs are being actively translated is the Translating Ribosome Affinity Purification (TRAP) methodology, which was developed by Nathaniel Heintz at Rockefeller University. TRAP does not involve ribosome footprinting but provides cell type-specific information.

<span class="mw-page-title-main">Slippery sequence</span>

A slippery sequence is a small section of codon nucleotide sequences that controls the rate and chance of ribosomal frameshifting. A slippery sequence causes a faster ribosomal transfer which in turn can cause the reading ribosome to "slip." This allows a tRNA to shift by 1 base (−1) after it has paired with its anticodon, changing the reading frame. A −1 frameshift triggered by such a sequence is a programmed −1 ribosomal frameshift. It is followed by a spacer region, and an RNA secondary structure. Such sequences are common in virus polyproteins.

<span class="mw-page-title-main">Translation regulation by 5′ transcript leader cis-elements</span>

Translation regulation by 5′ transcript leader cis-elements is a process in cellular translation.

References

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  10. 1 2 Darnell AM, Subramaniam AR, O'Shea EK (July 2018). "Translational Control through Differential Ribosome Pausing during Amino Acid Limitation in Mammalian Cells". Molecular Cell. 71 (2): 229–243.e11. doi:10.1016/j.molcel.2018.06.041. PMC   6516488 . PMID   30029003.
  11. 1 2 Buchan JR, Stansfield I (September 2007). "Halting a cellular production line: responses to ribosomal pausing during translation". Biology of the Cell. 99 (9): 475–87. doi: 10.1042/BC20070037 . PMID   17696878.
  12. ↑ Collart MA, Weiss B (February 2020). "Ribosome pausing, a dangerous necessity for co-translational events". Nucleic Acids Research. 48 (3): 1043–1055. doi:10.1093/nar/gkz763. PMC   7026645 . PMID   31598688.
  13. ↑ Chan, KH; Petrychenko, V; Mueller, C; Maracci, C; Holtkamp, W; Wilson, DN; Fischer, N; Rodnina, MV (14 August 2020). "Mechanism of ribosome rescue by alternative ribosome-rescue factor B." Nature Communications. 11 (1): 4106. Bibcode:2020NatCo..11.4106C. doi:10.1038/s41467-020-17853-7. PMC   7427801 . PMID   32796827.
  14. ↑ Carbone, Christine E.; Demo, Gabriel; Madireddy, Rohini; Svidritskiy, Egor; Korostelev, Andrei A. (December 2020). "ArfB can displace mRNA to rescue stalled ribosomes". Nature Communications. 11 (1): 5552. Bibcode:2020NatCo..11.5552C. doi:10.1038/s41467-020-19370-z. PMC   7641280 . PMID   33144582.
  15. ↑ Wolin SL, Walter P (November 1988). "Ribosome pausing and stacking during translation of a eukaryotic mRNA". The EMBO Journal. 7 (11): 3559–69. doi:10.1002/j.1460-2075.1988.tb03233.x. PMC   454858 . PMID   2850168.
  16. ↑ Manjunath H, Zhang H, Rehfeld F, Han J, Chang TC, Mendell JT (December 2019). "Suppression of Ribosomal Pausing by eIF5A Is Necessary to Maintain the Fidelity of Start Codon Selection". Cell Reports. 29 (10): 3134–3146.e6. doi:10.1016/j.celrep.2019.10.129. PMC   6917043 . PMID   31801078.
  17. ↑ Somogyi P, Jenner AJ, Brierley I, Inglis SC (November 1993). "Ribosomal pausing during translation of an RNA pseudoknot". Molecular and Cellular Biology. 13 (11): 6931–40. doi:10.1128/mcb.13.11.6931. PMC   364755 . PMID   8413285.
  18. ↑ Lopinski, John D.; Dinman, Jonathan D.; Bruenn, Jeremy A. (2000). "Kinetics of Ribosomal Pausing during Programmed −1 Translational Frameshifting". Molecular and Cellular Biology. 20 (4): 1095–1103. doi:10.1128/mcb.20.4.1095-1103.2000. PMC   85227 . PMID   10648594.
  19. ↑ Buskirk AR, Green R (March 2017). "Ribosome pausing, arrest and rescue in bacteria and eukaryotes". Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 372 (1716): 20160183. doi:10.1098/rstb.2016.0183. PMC   5311927 . PMID   28138069.
  20. ↑ Brar GA, Weissman JS (November 2015). "Ribosome profiling reveals the what, when, where and how of protein synthesis". Nature Reviews. Molecular Cell Biology. 16 (11): 651–64. doi:10.1038/nrm4069. PMC   5522010 . PMID   26465719.
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