Translation
Paul Pumpens in Single-Stranded RNA Phages, 2020
Furthermore, the crystal structures of two complexes between recombinant MS2 capsids and RNA operator fragments have been determined at the 2.7 Å resolution (Valegård et al. 1997). The three-dimensional studies clearly confirmed the intrinsic role of the adenines A−10, A−7, A−4 and a pyrimidine at position −5. The RNA stem-loop, as bound to the protein, formed a crescent-like structure and interacted with the surface of the β-sheet of a coat protein dimer (for more detail, see Chapter 21). It made protein contacts with seven phosphate groups on the 5′ side of the stem-loop, with a pyrimidine base at position −5, which stacked onto a tyrosine, and with two exposed adenine bases, one in the loop and one at a bulge in the stem. The replacement of the wild-type uridine with a cytosine at position −5 increased the affinity of the RNA to the dimer significantly. The complex with RNA stem-loop having cytosine at this position differed from that of the wild-type complex mainly by having one extra intramolecular RNA interaction and one extra water-mediated hydrogen bond (Valegård et al. 1997). Figure 16.7 presents the actual secondary structures of the two RNA operators within the complex I.
Orders Norzivirales and Timlovirales
Paul Pumpens, Peter Pushko, Philippe Le Mercier in Virus-Like Particles, 2022
The x-ray crystallography led to a real breakthrough in understanding the protein-RNA interactions that were occurring during the RNA phage translational repression and genome encapsidation. Thus, the first crystal structure of a complex of recombinant MS2 capsids with the 19-nucleotide RNA operator was resolved at 2.7 Å (Valegård et al. 1994b, 1997; Stockley et al. 1995). Then, the crystal structures of the MS2 VLPs complexed with the RNA aptamers, which differed by their secondary structure from wild-type RNA (Convery et al. 1998; Rowsell et al. 1998; Grahn et al. 1999) or involved the presence of 2′-deoxy-2-aminopurine at the critical -10 position of the operator (Horn et al. 2004) were resolved. Further, the structure of the coat protein complexed with the operator RNA fragments were also solved by x-ray crystallography for the phages PP7 (Chao et al. 2008), PRR1 (Persson et al. 2013), and Qβ (Rumnieks and Tars 2014). Although the overall binding mode of the stem-loop to the coat was similar in all the studied cases, the details were surprisingly different among different phages. However, it should be noted here that all attempts to identify analogous coat-RNA interactions in the acinetophage AP205 and caulophage φCb5 failed until now (Kaspars Tārs, unpublished observations), suggesting that mechanisms of genome recognition and translational repression might differ significantly among the distant levivirus members. Figure 25.8 compares the different binding modes of the coat dimer-operator complexes of the phages MS2, Qβ, and PP7.
Molecular Farming Antibodies in Plants: From Antibody Engineering to Antibody Production
Maurizio Zanetti, J. Donald Capra in The Antibodies, 2002
a nuclease inhibitor to the translation mixture. Different constructs were designed to test requirements for optimal transcription efficiency, transcript stability, translation efficiency and protein folding. Optimal constructs contained a 5'-stem-loop (from the upstream region of the T7 phage gene 10), a 3'-stem-loop (from a modified E. coli lipoprotein terminator) and a 116 amino acid spacer (derived from gene III of bacteriophage M13) fused to the C-terminus of the scFv. The stem-loops at both ends of the transcript protect against degradation by exonucleases and though stabilize the mRNA. A spacer was fused to the C-terminus of the protein to allow the protein to completely emerge from the ribosome and to give it sufficient distance not to interfere with protein folding. With the optimized system it was possible to select a specific scFv from a mixture of 1 specific scFv mRNA in 108 mRNAs encoding a non-specific scFv. Recently the system has been successfully used by Hanes et al. [177] to select and evolve high-affinity antibodies from a diverse library. Variants of a selected scFv showed a 65-fold higher affinity to the antigen than the likely scFv progenitor.
The vital role of animal, marine, and microbial natural products against COVID-19
Published in Pharmaceutical Biology, 2022
Aljawharah A. Alqathama, Rizwan Ahmad, Ruba B. Alsaedi, Raghad A. Alghamdi, Ekram H. Abkar, Rola H. Alrehaly, Ashraf N. Abdalla
The 5′ cap end of the viral genome has a leader series and untranslated region (UTR) composed of multiple regions. These are crucial to the formation of the many stem loop structures that are necessary for RNA replication and transcription. At the accent gene there are transcriptional regulatory sequences (TRSs) composed of a specific portion of 50–100 nucleotides required for the expression of each of those genes. The RNA structures needed to replicate and synthesize RNA are located in the 3′ UTR. The two-third (20 kilobases) of the genome consists of replicase genes known as open reading frames 1a and ab (ORF1ab), and encoded non-structural proteins (nsp), whereas the remaining region of the total viral genome (10 kilobases) encodes structural and accent proteins such as structural proteins involving spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Furthermore, the structural genes such as ORF3a, ORF3d, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF14, and ORF10 genes encode nine accessory proteins. The CoV genome is structured in the following order: 5′-leader-UTR-ORF-S-E-M-N-accessory proteins genome-3′ UTR-poly (A) tail with accessory genes interspersed among the structural genes at the 3′ end of the genome (Pal et al. 2020; Yadav et al. 2021).
COVID-19: a wreak havoc across the globe
Published in Archives of Physiology and Biochemistry, 2023
Heena Rehman, Md Iftekhar Ahmad
The genome of coronavirus is positive-sense, non-segmented, linear RNA of 28–32 kilobase (Lee et al. 1991, Bonilla et al.1994, Drosten et al. 2003). The genome is helically coiled and is encapsulated by nucleocapsid (N) protein. The genome consists of 5′ cap structure and 3′ poly adenine (A) tail which facilitates in acting as mRNA for translation of polyproteins (Van Marle et al. 1995). The replicase gene comprises around 20 kilobase of the genome and encodes non-structural proteins. The 5′ end of RNA consists of leader sequence and untranslated region that contains multiple stem-loop structure. This multiple stem lop structure is required for replication and transcription. The 3′ (untranslated region) UTR is essential for the replication and synthesis of viral RNA. The gene in coronavirus is organised as 5′leader-UTR-replicase-S-E-M-N-3’UTR-poly(A) tail (Figure 2). The accessory proteins are not important for in vitro replication, but they play a significant role in the pathogenesis (Zhao et al. 2012).
Challenges with the discovery of RNA-based therapeutics for flaviviruses
Published in Expert Opinion on Drug Discovery, 2023
Mei-Yue Wang, Rong Zhao, Yu-Lan Wang, De-Ping Wang, Ji-Min Cao
Recent studies have revealed the structures that are critical for the replication of flaviviruses and may provide insights into the design of antiviral drugs to fight against flaviviruses. For example, at the 5’ end of the RNA genome of flaviviruses, there is a ~ 70 nucleotide stem-loop structure called stem-loop A (SLA), which promotes RNA synthesis. The non-structural protein 5 (NS5) of the flavivirus specifically recognizes SLA to activate the synthesis of RNA and the methylation of the 5’ guanosine cap [150]. Another study showed that the endoplasmic reticulum-localized RNA-binding proteins (RBPs), ribosome-binding protein 1 (RRBP1), and vigilin, can directly bind to viral RNA and play a role in the distinct stages of the life cycle of flaviviruses, providing an RNA-centric perspective on the viral infection [151]. It can be inferred that RNA-based candidates that target SLA, RRBP1, and vigilin may interfere with the life cycle of flaviviruses and therefore have the potential to function as antivirals.