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With the genes bound in a nucleus, the eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its mRNA from degrading before it is translated. Eukaryotes also employ three different polymerases that each transcribe a different subset of genes.
Eukaryotic mRNAs are usually monogenic , meaning that they specify a single protein. Unlike the prokaryotic polymerase that can bind to a DNA template on its own, eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then to help recruit the appropriate polymerase.
The features of eukaryotic mRNA synthesis are markedly more complex than those of prokaryotes. Instead of a single polymerase comprising five subunits, the eukaryotes have three polymerases that are each made up of 10 subunits or more.
Each eukaryotic polymerase also requires a distinct set of transcription factors to bring it to the DNA template.
The rRNA molecules are considered structural RNAs because they have a cellular role but are not translated into protein. The rRNAs are components of the ribosome and are essential to the process of translation. Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation. RNA polymerase II is responsible for transcribing the overwhelming majority of eukaryotic genes. The toxin prevents the enzyme from progressing down the DNA, and thus inhibits transcription.
Knowing the transcribing polymerase can provide clues as to the general function of the gene being studied. Because RNA polymerase II transcribes the vast majority of genes, we will focus on this polymerase in our subsequent discussions about eukaryotic transcription factors and promoters.
Eukaryotic promoters are much larger and more intricate than prokaryotic promoters. However, both have a sequence similar to the sequence of prokaryotes.
This sequence is not identical to the E. The thermostability of A—T bonds is low and this helps the DNA template to locally unwind in preparation for transcription. Transcription factors that bind to the promoter are called basal transcription factors. The other transcription factors systematically fall into place on the DNA template, with each one further stabilizing the pre-initiation complex and contributing to the recruitment of RNA polymerase II.
Basal transcription factors are crucial in the formation of a preinitiation complex on the DNA template that subsequently recruits RNA polymerase II for transcription initiation. The complexity of eukaryotic transcription does not end with the polymerases and promoters. An army of other transcription factors, which bind to upstream enhancers and silencers, also help to regulate the frequency with which pre-mRNA is synthesized from a gene. Enhancers and silencers affect the efficiency of transcription but are not necessary for transcription to proceed.
Multiple studies have demonstrated that only the intact RING domain is essential for the inhibitory activity of the Z protein mediated by direct binding to L 11 , 16 , 17 , However, the molecular mechanism of inhibition remains unclear because of a lack of structural information on the L—Z complex. The structure illustrates the negative regulation mechanism of arenavirus L protein through interaction with the Z protein.
Further attempts to express and purify the Z protein in Escherichia coli as a fusion to the C-terminus of maltose-binding protein MBP yielded sufficient monomeric MBP-Z for structural and biochemical studies Supplementary Fig. However, most of the fusion protein formed aggregates. The interdomain borders of each protein are labeled with residue numbers. Molecular weights in kilodaltons of marker are shown on the left, and bands are labeled on the upper and right.
The data shown are representative results of more than two independent experiments. The density map is assembled from the individual domains of L and the RING domain of Z, indicated and colored as in a.
The reference-free 2D class averages indicated two forms of particles in the cryo-EM dataset resembling the monomeric and dimeric MACV L protein, respectively. After further data processing, the two structures were reconstructed to overall resolutions at 4.
The final density maps for the monomeric and dimeric L—Z complexes were highly similar to the apo MACV L protein structure 15 , except that additional densities were observed that were designated as Z protein in subsequent model building Fig.
This result was consistent with previous studies suggesting that the Z protein might bind to L in its monomer form or at a molar ratio of Structural comparison revealed that the monomeric L—Z complex was nearly identical to one monomer of the dimeric complex Supplementary Fig. The structure of the L protein in the monomeric and dimeric L—Z complexes was also highly similar to those of apo L protein monomer and dimer Fig.
Considering the high similarity of the monomeric and dimeric L—Z complexes, only the monomeric complex is discussed below. The domains are indicated and colored as in Fig. Side chains of conserved cysteines and histidine involved in Zn binding are shown as sticks and labeled. Although full-length MBP-Z fusion protein is used for structural study, only residues 34—82 are visible in the complex.
Residues involved in the interaction with the L protein are marked below with black solid triangle, while conserved cysteines and histidine involved in Zn binding are marked below with green italicized C or H. Secondary structural elements are depicted on the top of the alignments. The N- and C-termini were flexible 9 , 10 and not resolved in this study.
This result agrees with previous binding experiments 16 , 17 , which also suggested that the N- and C-termini of Z might not be essential for its inhibitory effects in vitro In comparison, the last turn of the helix in LASV Z residues 58—61 in the NMR structure seems to unfold, suggesting that it could offer more flexibility to the C-terminus.
Loop 1 of the Z protein contacts the L protein core lobe and the palm domain, while loop 2 interacts with the core lobe and the thumb ring Fig. The side chains of zTrp43 and zPhe44 are inserted into this hydrophobic pocket, just like one end of the staple Fig.
In addition to this hydrophobic interaction, Z Arg36 hydrogen bonds to the main chain of L Asn and the side chain of L Thr from the palm domain of of the RdRp using main chain and side chain, respectively. Inset zoom, the Z protein is represented as ribbon with the two binding sites outlined with black boxes and shown in b.
Left, loop 1 of Z interaction with palm and core lob of the L protein; Right, loop 2 interaction with core lob and thumb ring. The backbones of L and Z are in ribbons representation. Side chains and main chains involved in interactions are shown as sticks and labeled. Oxygen, nitrogen and sulfur atoms shown in red, blue and yellow, respectively, and salt bridges and hydrogen bonds are indicated as black dashed lines.
The catalytic site is indicated by a red asterisk and the CTD is is represented by a gray oval. The hidden product exit tunnel is showed as red dashed line. Sequence alignment of multiple Arenaviridae family Z proteins indicated that among the residues involved in the interaction between the Z and L proteins, the residues interacting with L in loop 1 are more conserved than those in loop 2 Supplementary Fig. In loop 1 of the Z protein, Trp43 is totally conserved in all arenaviruses, while Phe44 is conserved in most arenaviruses and Arg36 is conserved in all the New World arenaviruses.
Other mutants showed almost no effect on the interaction Supplementary Fig. The result is in consistence with the conservation of key residues according to the sequence alignment.
This finding is consistent with the results of a previous study, implying that Z protein cross-inhibits more closely related arenavirus and cannot function as broadly active inhibitors Thus, the interaction pattern between the L and Z proteins is probably universal among members of the Arenaviridae family, although the interaction details may be specific to individual arenavirus clades as a result of independent evolution.
Previous structural study showed that there was no large conformational rearrangement between apo and vRNA-bound MACV L protein, especially in regions required for Z binding It implies that the Z protein might interact with L in multiple structural or functional states, such as the monomeric and dimeric L protein in both apo and vRNA-bound states.
Binding and inhibiting the multiple states of the L protein increase the chance that Z functions the polymerase inhibitory activities, especially when the Z protein under low concentration in vivo. It might be a mechanism for the Z protein to improve the efficiency of inhibition. It indicates that the functions of Z mediated by RING domain are likely mutually exclusive because of steric hindrance in these three states. From the proposed model, the Z protein in the MACV L—Z complex is proximal to the putative product RNA exit surrounded by the structural elements of core lobe, palm domain and thumb ring, about Considering domain rearrangement is needed for sNSV RNA polymerases to perform and coordinate complex catalytic reactions 6 , 19 , 20 , 21 , 22 , the Z protein might exert the inhibitory function by locking the multiple domains of L in a catalytically inactive state.
The polymerase activity of L is inhibited via the blocking of product RNA extension. The flexible N-terminus, especially 10 residues adjacent RING domain 16 , is able to approach a helix residues — of palm domain and two loops residues — and — of thumb ring, which participate in the formation of the putative product exit tunnel.
It might be possible that the N-terminus of Z may also play a role in inhibiting RNA synthesis by blocking the putative product exit tunnel of L. The results and conclusion are basically the same, while differences are not excluded. Limited by convincing evidence, no consensus is reached to explain the inhibitory mechanism of Z and therefore further studies are needed.
Effective countermeasures against arenaviruses, such as vaccines and antiviral drugs, are not currently available The unique strategy used by arenavirus Z protein to inhibit the activity of L provides a novel target for the development of inhibitors.
Elucidation of the structural details and molecular mechanisms underlying the L—Z complex formation will be instrumental for structure-based development of inhibitors against arenaviruses, such as engineered Z proteins that more potently inhibit the L protein. Sf9 and Hi5 insect cells Invitrogen were used for baculovirus propagation and protein expression. After washing three times with Lysis Buffer to remove non-specifically bound protein, the target protein was subsequently eluted by 2.
However, the monomeric MBP-Z fusion protein was purified to homogeneity for structural study. Each image was exposed for 6. Each movie stack was binned and then subjected to beam-induced motion correction and anisotropic magnification correction by the software MotionCor2 This procedure generated two summed images with or without dose weighting for each movie stack.
Further cryo-EM data processing were performed in Relion Particles were first automatic picking using Gautomatch by K. Zhang and processed with reference-free 2D classification. After the first round of 2D classification, monomeric and dimeric particles were separated, re-extracted with adjusted box-size and subjected to further 2D classification individually. For the monomeric L—Z complex, five 2D class average images of the monomeric L—Z complex were selected as templates for automatic particle picking against the entire dataset.
A total of 1,, particles were picked in micrographs and processed by reference free 2D classification. One among the five classes containing , particles were selected for further 3D auto-refinement, which resulted in a 4. For the dimeric L—Z complex, three 2D class average images of the dimeric L—Z complex were selected as templates for automatic particle picking and 1,, particles were picked.
The best class among the three containing , particles were selected for further 3D auto-refinement and resulted in a 4. For monomer, 1,, particles were picked in micrographs and , monomeric particles were selected for 3D auto-refinement and resulted in a 3. For dimer, 1,, particles were picked and 52, dimeric particles were selected for 3D auto-refinement and resulted in a 5. The local resolutions of the final maps were calculated using ResMap Statistics for image collection and processing are summarized in Supplementary Table 1.
The model of the L—Z complex was improved by a few iterative rounds of manual modification in Coot and real-space refinement using PHENIX 36 until no further improvement could be obtained.
The models were refined iteratively by cycles of real-space refinement using PHENIX and manual modification in Coot until no further improvement could be obtained.
In part, this is because it is difficult to infer exactly where a eukaryotic promoter begins and ends. Some promoters occur within genes; others are located very far upstream, or even downstream, of the genes they are regulating. However, when researchers limited their examination to human core promoter sequences that were defined experimentally as sequences that bind the preinitiation complex, they found that promoters evolve even faster than protein-coding genes.
It is still unclear how promoter evolution might correspond to the evolution of humans or other higher organisms. However, the evolution of a promoter to effectively make more or less of a given gene product is an intriguing alternative to the evolution of the genes themselves. These sequences alone are sufficient for transcription initiation to occur, but promoters with additional sequences in the region from to upstream of the initiation site will further enhance initiation.
Genes that are transcribed by RNA polymerase III have upstream promoters or promoters that occur within the genes themselves. Eukaryotic transcription is a tightly regulated process that requires a variety of proteins to interact with each other and with the DNA strand. Although the process of transcription in eukaryotes involves a greater metabolic investment than in prokaryotes, it ensures that the cell transcribes precisely the pre-mRNAs that it needs for protein synthesis. As discussed previously, RNA polymerase II transcribes the major share of eukaryotic genes, so this section will focus on how this polymerase accomplishes elongation and termination.
Although the enzymatic process of elongation is essentially the same in eukaryotes and prokaryotes, the DNA template is more complex. When eukaryotic cells are not dividing, their genes exist as a diffuse mass of DNA and proteins called chromatin. The DNA is tightly packaged around charged histone proteins at repeated intervals. These DNA—histone complexes, collectively called nucleosomes, are regularly spaced and include nucleotides of DNA wound around eight histones like thread around a spool.
For polynucleotide synthesis to occur, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome. The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,—2, nucleotides beyond the end of the gene being transcribed. Genes transcribed by RNA polymerase I contain a specific nucleotide sequence that is recognized by a termination protein. Improve this page Learn More.
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