27.3.1 Regulation of Translation
Heterogeneity of Ribosome Structure
Over the years, many studies performed in eukaryotes presented evidence that ribosomes can vary in their protein and rRNA complement between different cell types and developmental states. These observations culminated in the postulation of the ‘ribosome filter hypothesis’ by Mauro and Edelman in the year 2002. The authors propose that the ribosome composition functions as translation determination factor. Depending on the RPs and rRNA sequences represented in the respective ribosome, the complex acts like a filter that selects for specific mRNAs and hence modulates translation (Fig. 27.3.1). RP heterogeneity can arise from differential expression of paralogs/homologs of RP proteins within different cell types or occur due to differential post-translational modifications of RPs, such as phosphorylation. The protein to rRNA ratio may also slightly vary within ribosomal composition affecting translation efficiency and selectivity.
rRNA genes are also present in multiple copies throughout the genomes of organisms from all domains of life. For example, the bacteria Streptomyces coelicolor harbors six copies of divergent large subunit (LSU) rRNA genes that constitute at least five different LSU rRNA species in a cell. These genes were shown to be differentially transcribed during the morphological development of the organism. Similarly, B. subtilis harbors ten rRNA operons and their reduction to one copy increased the doubling time as well as the sporulation frequency and the motility of the resulting mutant.
Modification of the rRNA also provides another avenue of ribosomal heterogeneity. Similar to tRNA, rRNA residues can be chemically modified and commonly have 2-OH methylation. The conversion of uridine to psuedoruridine is also quite common. In eukaryotes, the modifications are facilitated by snoRNAs and their tissue specific expression might be a source for ribosome specialization. In light of the increasing evidence, ribosome heterogeneity, though still far from being entirely understood, proves to be an integral mechanism to modulate and fine tune protein synthesis in response to environmental signals in all organisms.
Effects of Sequence and Secondary Structure in mRNA
The amount of protein produced from any given mRNA (i.e., the translational output) is influenced by multiple factors specified by the primary nucleotide sequence. These factors include GC content, codon usage, codon pairs, and secondary structure.
For example, 5’UTR sequences in the mRNA may interact with small miRNAs and lead to RNA interference. miRNA interactions may also target mRNA for degradation (Figure 27.3.2). This process is aided by protein chaperones called argonautes. This antisense-based process involves steps that first process the miRNA so that it can base-pair with a region of its target mRNAs. Once the base pairing occurs, other proteins direct the mRNA to be destroyed by nucleases. Fire and Mello were awarded the 2006 Nobel Prize in Physiology or Medicine for this discovery.
Figure modified from: Wikimedia Commons
Effects of the Nascent Peptide on Ribosome Efficiency
Since the Peptidyl Transferase Center (PTC) is buried within the large subunit, during translation the nascent peptide chain (NC) exits through a 100 Å long tunnel (Figure 27.3.3). The exit tunnel plays an active role in protein synthesis. Certain peptide sequences specifically interact with tunnel walls and induce ribosome stalling. Furthermore, the exit tunnel is a binding site for a clinically important class of antibiotics known as the macrolides.
When synthesizing proteins containing proline stretches (i.e. several prolines in a row), ribosomes become stalled. Stalling is alleviated by a specialized elongation factor, EF-P in bacteria. Recently, cryo-EM structures of a ribosome stalled by a proline stretch with and without EF-P were resolved. In simulations of the PTC region, elongation factor P (EF-P) was observed to stabilize the P-site tRNA in a conformation compatible with peptide bond formation, while in the absence of EF-P, the P-site tRNA moved away from the A-site tRNA.
The exit tunnel can accommodate 30–60 AAs, depending on the level of NC compaction. The rate of translation of about 4–22 AA per second in bacteria provides the NC with sufficient time to explore its conformational space and to start folding when still bound to the ribosome-tRNA complex.
The 26S proteasome is the central element of proteostasis regulation in eukaryotic cells. It is required for the degradation of protein factors in multiple cellular pathways and it plays a fundamental role in cell stability. The main aspects of proteasome-mediated protein degradation have been largely described during the last three decades with the use of intense cellular, molecular, structural and chemical biology research and tool development. The 26S proteasome has a structural configuration that confines the proteolytic active sites in a location unreachable for native and functional proteins, thus preventing uncontrolled degradation. The proteolytic active sites are found in the interior of a barrel-shaped core particle (CP or 20S). The entrances of the tunnel, placed at the distal ends of the barrel, are commonly occupied by the regulatory particle (RP or 19S), a sophisticated protein assembly that acts as a substrate processing machine. the regulatory particle has the important role of receiving, deubiquitinating, unfolding and translocating substrates to the CP and it adopts different configurations depending on the activity states they exhibit. This process typically requires ATP hydrolysis. Moreover, conformationally distinct proteasomes may show different subcellular distributions depending on functional requirements in each cellular type and environmental situations. Proteasomes are distributed throughout the cell, detected in the cytoplasm and in the nucleus, and they can localize to hotspots in distinct intracellular regions or specific sites with high protein metabolism or with specific protein degradation requirements.
Proteins are normally targeted to the proteasome by means of ubiquitin labels attached covalently to a lysine residue, usually in a chained form that produces a polyubiquinated protein. The process of protein polyubiquitination is carried out by a highly specialized and diverse enzymatic system, which
includes families of ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3).
The covalent attachment of ubiquitin to specific target proteins is mainly accomplished by stepwise enzymatic cascade reactions, and ubiquitin is attached to the substrates via the concerted action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). The attachment of ubiquitin or ubiquitin chains to the substrate is a successive process (Figure 27.3.5). First, the C-terminal carboxylic acid is activated by adenylation using a molecule of ATP forming an adenylate (AMP-) intermediate. The adenylate acts as a good leaving group during the next reaction where an E1-ubiquitin thioester bond is formed between the C-terminal Gly carboxyl group of ubiquitin and the active site Cys of the E1 enzyme. AMP leaves the active site at this point. Note that ATP is used in many reactions to activate carboxylic acid functional groups through the formation of an adenylate intermediate and that this will be seen as a theme in many different types of reactions throughout this textbook. Once the ubiquitin is docked as a thioester on E1, it can be transferred to a Cys residue of the E2 enzyme to form an E2-ubiquitin thioester-linked intermediate. This enzymatic reaction is known as a transesterification.
Figure from: Hann, Z.S., et. al. (2019) PNAS 116(31)15475-15484.
Eventually the E2 transfers the ubiquitin to the substrate protein by E3. Ubiquitin is conjugated to the target protein through an isopeptide bond between its C-terminal glycine (Gly76) and the ε-amino group of a lysine residue. There are three typical ways of linking the ubiquitin with the substrate (Figure 27.3.6). The first is called mono-ubiquitination, which refers to the modification of one site of the modification of a substrate by a single ubiquitin molecule. The second is multi-mono-ubiquitination, which means adding several ubiquitin molecules repetitively to distinct sites (multi-mono-ubiquitination). The third is called polyubiquitination (including linear polyubiquitination and branched polyubiquitination), in which ubiquitin molecules are added to the same site (polyubiquitination, including linear polyubiquitination and branched polyubiquitination) of a substrate. In the second and third ways of linking, the previously attached ubiquitin serves as the “acceptor” of subsequently added ubiquitin. Of course, polyubiquitin chains linked by the same Lys are homogeneous, while those linked at different Lys are heterogeneous or mixed ones.
Figure from: Liu, W., et.al. (2020) Int J Mol Sci 21(8):2894
Subsequently, the substrate complex tagged by the ubiquitin is either degraded by the 26S proteasome or executes nonproteolytic functions, such as the regulation of gene expression, cellular trafficking, or other biological function. In most cases, polyubiquitinated proteins are recognized and degraded by the 26S proteasome, and the ubiquitin or ubiquitin chain is hydrolyzed and freed by deubiquitinating enzymes (DUBs) for reuse in further conjugation cycles after being removed from the substrate protein (Figure 27.3.6).
The family of E3 enzymes is large and diverse. It is estimated that there are 600-700 E3 enzymes in humans, representing approximately 5% of the human genome. Thus, E3 enzymes can be very substrate specific, leading to the specialized degradation of a small subset of proteins within the cell. E2 enzymes are the intersection between E1 and E3 enzymes and help to determine the ubiquitination of specific target proteins by interacting with different types of E3 enzymes.
This chapter was remixed and adapted from the following resources under creative commons licensing:
- Wikipedia contributors. (2020, June 27). Wobble base pair. In Wikipedia, The Free Encyclopedia. Retrieved 16:47, August 12, 2020, from https://en.Wikipedia.org/w/index.php?title=Wobble_base_pair&oldid=964760055
- Lorenz, C., Lünse, C., and Mörl, M. (2017) tRNA modifications: Impact on structure and thermal adaptation. Biomolecules 7(2)35. Available at: https://www.mdpi.com/2218-273X/7/2/35/htm
- Pan, T. (2018) Modifications and functional genomics of human transfer RNA. Cell Research 28:395-404. Available at: https://www.nature.com/articles/s41422-018-0013-y#Sec3
- Bednárová, A., Hanna, M., Durham, I., Van Cleave, T., England, A., Chaudhuri, A., and Krishnan, N. (2017) Lost in translation: Defects in transfer RNA modifications and neurological disorders. Front. Mol Neurosci. 10:135. Available at: https://www.researchgate.net/publication/316440980_Lost_in_Translation_Defects_in_Transfer_RNA_Modifications_and_Neurological_Disorders
- Wikipedia contributors. (2020, May 17). Transfer RNA. In Wikipedia, The Free Encyclopedia. Retrieved 23:03, August 15, 2020, from https://en.Wikipedia.org/w/index.php?title=Transfer_RNA&oldid=957227343
- Gomez, M.A.R., and Ibba M. (2020) Aminoacyl-tRNA Synthetases. RNA, doi: 10.1261/rna.071720.119 Available at: https://rnajournal.cshlp.org/content/early/2020/04/17/rna.071720.119.abstract
- Li, R., Macnamara, L.M., Leuchter, J.D., Alexander, R.W., and Cho, S.S. (2015) MD Simulations of tRNA and Aminoacyl-tRNA Syntetases: Dynamics, Folding, Binding, and Allostery. Int. J. Mol Sci. 16(7):15872-15902. Available at: https://www.mdpi.com/1422-0067/16/7/15872
- Wikipedia contributors. (2020, August 15). Ribosome. In Wikipedia, The Free Encyclopedia. Retrieved 05:22, August 16, 2020, from https://en.Wikipedia.org/w/index.php?title=Ribosome&oldid=973144885
- Kater, L., Thoms, M., Barrio-Garcia, C., Cheng, J., Ismail, S., Ahmed, Y.L., Bange, G., Kressler, D., Berninghausen, O., Sinning, I., Hurt, E., and Beckmann, R. (2017) Visualizing the assembly pathway of nucleolar Pre-60S Ribosomes. Cell 171(7):1599-1610. Available at: https://www.sciencedirect.com/science/article/pii/S0092867417314290
- Bock, L.V., Kolár, M.H., Grubmüller, H. (2018) Molecular simulations of the ribosome and associated translation factors. Cur. Op. Struc. Bio. 49:27-35. Available at: https://www.sciencedirect.com/science/article/pii/S0959440X1730132X
- Doris, S.M., Smith, D.R., Beamesderfer, J.N., Raphael, B.J., Nathanson, J.A., and Gerbi, S.A. (2015) Universal and domain-specific sequences in 23S-28S ribosomal RNA identified by computational phylogenetics. RNA 21:1719-1730. Available at: https://www.researchgate.net/publication/281141702_Universal_and_domain-specific_sequences_in_23S-28S_ribosomal_RNA_identified_by_computational_phylogenetics
- Aleksashin, M.A., Leppik, M., Hochenberry, A.J., Klepacki, D., Vázquez-Laslop, N., Jewett, M.C., Remme, J., and Mankin A.S. (2019) Assembly and functionality of the ribosome with tethered subunits. Nature Communications 10:930. Available at: https://www.nature.com/articles/s41467-019-08892-w#rightslink
- Wikipedia contributors. (2020, July 3). Formylation. In Wikipedia, The Free Encyclopedia. Retrieved 22:03, August 16, 2020, from https://en.Wikipedia.org/w/index.php?title=Formylation&oldid=965827476
- Gualerzi, C.O., and Pon C.L. (2015) Initiation of mRNA translation in bacteria: structural and dynamic aspects. Cell Mol Life Sci. 72:4341-4367. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4611024/
- Wikipedia contributors. (2020, June 14). Kozak consensus sequence. In Wikipedia, The Free Encyclopedia. Retrieved 06:03, August 18, 2020, from https://en.Wikipedia.org/w/index.php?title=Kozak_consensus_sequence&oldid=962444929
- Knight, J.R.P., Garland, G., Pöyry, T., Mead, E. Vlahov, N., Sfakianos, A., Frosso, S., De-Lima-Hedayioglu, F., Mallucci, G.R., von der Haar, T., Smales, C.M., Sansom, O.J., and Willis, A.E. (2020) Control of translation elongation in health and disease. Dis. Mod. and Mech. 13: dmm043208. Available at: https://dmm.biologists.org/content/13/3/dmm043208
- Adio, S., Sharma, H., Senyushkina, T., Karki, P., Maracci, C., Wohlgemuth, I., Holtkamp, W., Peske, R., and Rodina, M.V. (2018) Dynamics of ribosomes and release factors during translation termination in E. coli. eLife 7:e34253. Available at: https://elifesciences.org/articles/34252
- Ge, X., Oliveira, A., Hjort, K., Bergfors, T., Guitiérrez-de-Terán, H., Andersson, D.I., Sanyal, S., and Åqvist, J. (2019) Inhibition of translation termination by small molecules targeting ribosomal release factors. Scientific Reports 9: 15424. Available at: https://www.nature.com/articles/s41598-019-51977-1#rightslink
- Svidritskiy, E., Demo G., Loveland A.B., Xu, C., and Korosteleve, A.A. (2019) Extensive ribosome and RF2 rearrangements during translation termination. eLife 8:e46850. Available at: https://elifesciences.org/articles/46850
- Sauert, M., Temmel, H., and Moll, I. (2015) Heterogeneity of the translational machinery: Variations on a common theme. Biochimie 114:39-47. Available at: https://www.sciencedirect.com/science/article/pii/S0300908414003952
- Shen, Q., Wang, G., Li, S., Liu, X., Lu, S., Chen, Z., Song, K., Yan, J., Geng, L, Huang, Z., Huang, W., Chen, G., and Zhang, J. (2016) ASDv3.0: Unraveling allosteric regulation with structural mechanisms and biological networks. Nucleic Acids Research 44(D1):D527-D535. Available at: https://academic.oup.com/nar/article/44/D1/D527/2503129
- Wikipedia contributors. (2020, January 19). Isozyme. In Wikipedia, The Free Encyclopedia. Retrieved 03:42, May 19, 2020, from https://en.Wikipedia.org/w/index.php?title=Isozyme&oldid=936548836
- Wikipedia contributors. (2020, April 30). COX-2 inhibitor. In Wikipedia, The Free Encyclopedia. Retrieved 07:00, May 22, 2020, from en.Wikipedia.org/w/index.php?title=COX-2_inhibitor&oldid=954080651
- Clarkson, C.W. (2018) Major Side Effects of NSAIDs and COX-2 Selective Inhibitors. TUSOM Pharmwiki. Available at: http://tmedweb.tulane.edu/pharmwiki/doku.php/nsaid_side_effects?do=
- Santos, A.L. and Lindner, A.B. (2017) Protein Porsttranslational Modifications: Roles in Aging and Age-Related Disease. Oxidative Medicine and Cellular Longevity, Article ID: 5716409. Available at: https://www.hindawi.com/journals/omcl/2017/5716409/#copyright
- Wikipedia contributors. (2020, May 12). Protein phosphorylation. In Wikipedia, The Free Encyclopedia. Retrieved 17:38, May 25, 2020, from en.Wikipedia.org/w/index.php?title=Protein_phosphorylation&oldid=956228409
- Szylveszter, K.P., Németh, T., and Mócsai, A. (2019) Tyrosine Kinases in Autoimmune and Inflammatory Skin Diseases. Front. Immunol. 10.3389(2019.01862). Available at: https://www.frontiersin.org/articles/10.3389/fimmu.2019.01862/full
- Wikipedia contributors. (2020, May 7). Histone. In Wikipedia, The Free Encyclopedia. Retrieved 21:12, May 25, 2020, from en.Wikipedia.org/w/index.php?title=Histone&oldid=955458038
- Wikipedia contributors. (2020, May 13). Mucin. In Wikipedia, The Free Encyclopedia. Retrieved 01:10, June 7, 2020, from en.Wikipedia.org/w/index.php?title=Mucin&oldid=956387296
- Wikipedia contributors. (2020, March 15). Allosteric regulation. In Wikipedia, The Free Encyclopedia. Retrieved 04:07, June 7, 2020, from en.Wikipedia.org/w/index.php?title=Allosteric_regulation&oldid=945637073
- Dixit, Ajay. Dawra, Rajinder K. Dudeja, Vikas. Saluja, Ashok K. (2016). Role of trypsinogen activation in genesis of pancreatitis. Pancreapedia: Exocrine Pancreas Knowledge Base, DOI: 10.3998/panc.2016.25
- Coll-Martinez, B., and Crosas, B. (2019) How the 26S Proteasome Degrades Ubiquitinated Proteins in teh Cell. Biomolecules 9(9):395. Retrieved from: https://www.mdpi.com/2218-273X/9/9/395
- Liu, W., Tang, X., Qi, X., Ghimire, S., Ma, R., Li, S., Zhang, N., and Si H. (2020) The Ubiquitin Conjugating Enzyme: An Important Ubiquitin Transfer Platform in Ubiquitin-Proteosome System. Int J Mol Sci 21(8):2894. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215765/