The Single-Stranded DNA Binding Protein of Bacteriophage T4
James F. Kane in Multifunctional Proteins: Catalytic/Structural and Regulatory, 2019
Lagging strand synthesis can be studied in systems that utilize additional T4 proteins. A five protein replication system can use preprimed single-stranded DNA (like ΦX174) as a template. The five protein system includes the products of genes 32, 43, 44, 45, and 62. Several groups have reported that T4 DNA polymerase is stimulated by these other gene products to increase the rate of DNA polymerization and increase as well the processivity of the DNA replication complex.50,52,57,63–67 Some of the proteins in the replication complex may act to prevent the dissociation of DNA polymerase from the DNA template.65,67 The hydrolysis of ATP may be required for this reaction. Recent results suggest that this replication complex, in the presence of the ATP analogue ATP-γ-S, will not proceed through double-stranded DNA regions but is still capable of replicating single-stranded DNA.67 Most importantly, in the absence of the 32 protein or the 44, 45 and 62 proteins, DNA synthesis by DNA polymerase terminates at stable hairpin structures.66,67 The presence of gp32 or a mixture of gp44, gp45, and gp62 enhanced DNA synthesis through these regions. Interestingly, the addition of all four proteins to the same reaction is synergystically stimulatory.
Herpes Simplex Virus and Human CNS Infections
Sunit K. Singh, Daniel Růžek in Neuroviral Infections, 2013
In 1997, a model for HSV DNA replication was formulated (Boehmer and Lehman 1997). Once the β proteins are expressed, a number of proteins localize into the nucleus and assemble in DNA replication complexes at prereplicative sites, where viral DNA synthesis initiates on the circular molecule. Then, the UL9 (the origin binding) protein binds to specific elements—origin of replication (either OriL or Oris)—thus beginning to unwind the DNA. Then, it recruits ICP8 (the ssDNA binding protein) to the unwound ssDNA and they both recruit the five other viral replication proteins (helicase–primase complex of three proteins UL5, UL8, and UL52, viral DNA polymerase catalytic subunit UL30, and its processivity factor UL42) to begin the initial round of θ (theta) form replication (Wu et al. 1988). Leading strand synthesis involves the unwinding of the DNA and synthesis of a primer by the HSV helicase–primase complex. Then, replication switches from θ form to rolling circle mode, producing long head-to-tail concatemers of viral DNA by an unknown mechanism (Jacob et al. 1979). Concatemers are cleaved into monomeric molecules during packaging.
Experimental Protocols for Generation and Evaluation of Articular Cartilage
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi in Articular Cartilage, 2017
The use of random hexamers avoids the problems of 3′ bias seen with oligoDT primers during cDNA creation. OligoDT primers only create cDNA from mRNA species with 3′ poly A tails. This has limitations in that the processivity of reverse transcriptase may limit the length of cDNA created from long-sequence mRNAs. Generally, sequences exceeding 2 kb may have reduced representation in the final cDNA. Primers for RT-PCR that are used with OligoDT-generated cDNA should take this into account and preferably not be more than 1.5-2 kb from the 3′ end of the sequence. Furthermore, internal standards, such as 18S RNA, will not be produced unless random hexamers are used, as 18S RNA lacks the poly A tail.
Riboswitches as therapeutic targets: promise of a new era of antibiotics
Published in Expert Opinion on Therapeutic Targets, 2023
Emily Ellinger, Adrien Chauvier, Rosa A. Romero, Yichen Liu, Sujay Ray, Nils G. Walter
Since riboswitches are embedded near the 5’ end of mRNAs, they bind their respective ligand and change conformation while they are still being synthesized by RNAP [90], allowing for a dynamic response to a bacterial cell’s physiological conditions. Due to their narrow temporal window for gene regulation, riboswitches have the ability to interact with the nearby RNAP and its accessory proteins, as well as the pioneering ribosome, providing an underexplored mechanism to be exploited for the design of antibiotics. As a gateway for the regulation of transcription processivity, the positively charged RNA exit channel of RNAP constitutes an attractive platform in which nascent transcripts could establish key contact points to regulate the efficiency of RNA synthesis. Within the RNA exit channel, subdomains such as the ß-Flap or the Zinc Binding Domain (ZBD) have been found to participate in numerous regulatory pathways during the transcription cycle [91–96]. Even though the core RNAP structure is very well conserved in all domains of life, particular residues have been found only in bacteria [97] and could constitute a specific interface targeted by specific riboswitch features as well as future antibiotics. That is, going beyond directly targeting the ligand binding pocket, exploiting riboswitch interactions with the gene expression machinery presents a potentially fertile ground for novel drug design.
Molecular Diagnostic Tools for the Detection of SARS-CoV-2
Published in International Reviews of Immunology, 2021
Manali Datta, Desh Deepak Singh, Afsar R. Naqvi
The next target that enables the virus to manifest its infection is an RNA-dependent RNA polymerase [RdRp]. This protein is part of a multiprotein complex containing 500–600 amino acid residues embedded in the membrane. RdRp complex consists of an RNA polymerase, nsp12, whose processivity is augmented by nonstructural protein 7 [nsp7] and nsp8. The RdRp catalytic module generates full-length [–] RNA copies of the genome and uses it as a template for the synthesis of full-length [+] RNA genomes [13]. High error rates, typically about 1/34,000, in viral RdRp’s copying ensure enormous sequence variation in the RNA virus population, allowing rapid virus evolution under selective pressures imposed by the host immune response and/or drug treatments. Strand switching during RdRp copying is also a mechanism for RNA recombination, allowing RNA viruses to repair deleterious mutations, rearrange genes, and acquire new genes from other viruses or their host(s) further facilitating virus to evolve and mutate rapidly [14].
Molecular mechanisms governing axonal transport: a C. elegans perspective
Published in Journal of Neurogenetics, 2020
Amruta Vasudevan, Sandhya P. Koushika
Neurons have diverse cargo such as synaptic vesicles, mitochondria, endosomes, peroxisomes, lysosomes, and autophagosomes. These cargoes exhibit distinct mechanisms of transport regulation, often linked to their unique functions within neurons. The regulation of cargo transport occurs through a) initiation of cargo motility, b) processivity of movement, or c) halting of motion at specific locations. Signalling endosomes have been shown to trigger Dynein-mediated retrograde transport upon activation and endocytosis of Trk receptors (Maday et al., 2014). Synaptic vesicles, which often have to travel long distances to be delivered to distal presynaptic sites, regulate their processive motility by facilitating dimerization of the anterograde motor UNC-104/Kinesin-3 on the cargo surface (Klopfenstein, Tomishige, Stuurman, & Vale, 2002; Klopfenstein & Vale, 2004). Mitochondria, which have known roles in calcium homeostasis, are immobilized in axons at regions of high local calcium concentration through Miro, a calcium binding mitochondrial Rho GTPase that functions to recruit Kinesin-1 to the mitochondrial outer membrane (Guo et al., 2005). These studies collectively suggest that different neuronal cargo have evolved distinct cargo-specific mechanisms of transport regulation to promote their respective functions. Molecular mechanisms underlying cargo-specific regulation of transport have been covered extensively in the following review (Maday et al., 2014).
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