RNA polymerase (RNA polymerase): in a DNA chain or RNA template catalyzed by the nucleoside -5 '- triphosphate synthesis of RNA enzymes. Is catalyzed by the DNA as a template (template), ribose nucleoside triphosphate as the substrate, through the polymerization of the phosphodiester bond and the synthesis of RNA enzymes. Because the DNA in the cell's genetic information and gene transcription for the RNA are, therefore, also known as transcription.
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basic introduction to the structure function of the role of ring substitution effect start classification Research on Biological DNA polymerase RNA polymerase A comparison of DNA ligase. Template and enzyme II. Basic introduction to RNA polymerase transcription (RNA polymerase): in a DNA chain or RNA as a template catalyzed by the nucleoside -5 '- triphosphate synthesis of RNA enzymes. RNA polymerase catalyzed transcription of a number of protein subunits by the enzyme. Such as E. coli RNA polymerase composed of five subunits, the molecular weight of 480000, containing α, β, β ', δ, etc. 4 different peptides, where α is the two molecules, so the whole enzyme (holoenzyme) the composition of the is α2ββ'δ. RNA polymerase α subunit tetramer core (α2ββ ') in the formation; β subunit contains a nucleoside triphosphate binding site; β' subunit of DNA templates contain binding sites; and Sigma factor only and the RNA transcription initiation is related to the extension has nothing to do with the chain, once the transcription start, δ factor
to be released, and chain extension by the tetramer core enzyme (core enzyme) catalysis. Therefore, δ is to identify the role of transcription factors in the starting position, and to RNA polymerase binding site in the promoter. Bacterial RNA polymerase, DNA polymerase, like, like, have a very complex structure. Its active form (holoenzyme) for 15S, from 5 different types of polypeptide chain composition, arranged according to molecular size, respectively β '(155000), β (151000), σ (7000), α (36500) and ω (11000) . Each molecule has two RNA polymerase α subunit in addition, the remaining subunits are only one, so the whole enzyme β'βα2σω (450000) if the whole enzyme is spherical, you can calculate the diameter of about 100A, which is about double The 30bp DNA chain segment length. However, holoenzyme can be combined with about 60 nucleotides, suggesting that it should be oval shaped ball. σ subunit and the combination of the other peptide chain is not very strong. When the σ subunit from the holoenzyme, the remaining β'βα2ω called core enzyme. Catalytic core enzyme itself is able to inter-nucleotide phosphodiester bond formation, with or without the existence of rifampicin σ (rifampicin) and rifamycin (rifamycin) can be combined in the β subunit of this enzyme, where a strong inhibition. It inhibits RNA synthesis initiation rather than inhibit its extension.
RNA polymerase β subunit
rpoB mutations make the gene of rifampicin resistant bacteria. Streptavidin and the other an antibiotic bacteriolytic element (streptolydigin) can inhibit RNA chain extension, rpoB mutant cells but can also occur streptavidin bacteriolytic element resistance. On the whole β subunit appears to be the substrate of enzymes and nucleotide binding sites. Heparin competition with DNA and RNA polymerase combined. Heparin is a polyvalent anion sub, energy and β 'subunits, thereby inhibiting transcription in vitro. Shows β 'subunit of alkaline stronger for the combination of template DNA. α subunit function is not yet known. However, when the bacteriophage T4 infection of E.coli, its α subunit that is arginine ADP-ribose was based on. This modification of the RNA polymerase holoenzyme to its original identification of the promoter affinity decreased. So, some people think, α subunit function may identify the corresponding promoters. Why bacterial RNA polymerase structure needs such a large and complex molecular structure? And some phage-specific RNA polymerase is much smaller, only formed by a polypeptide chain. This proves that the institutions needed for RNA synthesis of the enzyme can be smaller than the host. This shows that the transcription of phage only a However, this enzyme can only recognize a few of the phage itself, the promoter of all; they can not identify the other promoter. For example, the bacteriophage T3 and T7 RNA polymerase molecules very similar, both are nothing but a polypeptide chain, molecular weight of about 11000
RNA polymerase
. They can quickly synthesized RNA. The rate at 37 ℃ up to about 200 nucleotides / sec. In contrast, host cell RNA polymerase transcription of the cell was able to many transcriptional units (> 1,000) in any one. Some of these transcription units by RNA polymerase transcription directly, without the help of other factors. But most of these transcription units only in the presence of additional protein factors in order to be the RNA polymerase by transcription. Therefore, it requires the host cell can imagine such a large and complex, at least in part reflects its need to interact with many other factors, rather than its inherent catalytic activity requirements. E. coli RNA polymerase subunits of the genes (in addition to ω subunit gene is still unknown outside) were present in three operons, the operon also contains the required protein synthesis and DNA synthesis genes. P71 major promoter (P) are in the left operon, RNA synthesis is to the right. Minor promoter (p), including a σ operon promoter (PHS, is only active under heat shock promoter), are located within the operon. These minor promoter only started in the right side of the gene. β operon in front of the two genes L11 and L1, is sometimes considered a separate operon formed, because another one before the L10 gene promoter. About 50 of these genes encode ribosomal proteins in some of the proteins. σ operon also contains the proteins required for replication, the DNA primase gene. It is not clear how these operons are regulated. σ operon has two consecutive promoter, the same as rRNA operons. Molecular
RNA polymerase by ppGpp
regulation, it is related with the growth rate. The genes of these operons to be transcribed is not equal to: σ and β in the beginning of the operon ribosomal protein gene behind the attenuator (attenuators), the RNA polymerase gene can then be greatly reduced transcription . Thus, in each cell the number of RNA polymerase subunit (about 3000) to be much less than the number of molecules of ribosomal protein (about 20,000), which is consistent with the needs of bacteria. However, paradoxically, is the DNA primase gene is located before the σ gene, however,
air force 1 high, the number of cells rather than the σ subunit primase more than 60 times the number of molecules (3000 more than 50). This may be because the mRNA encoded primase mRNA segments of the remaining part of the instability is much more than that. These are a few minor within the operon promoter,
nike air force 1, including a σ within operons can be activated by heat shock, indicating that the regulation of the actual and even more complex. The role of RNA polymerase (RNA polymerase) is the role of transcription RNA. Some RNA polymerase subunit a relatively complex structure. Such as E. coli RNA polymerase has four polypeptide chains, and another one for the synthesis of new RNA molecules σ factor, so it is the composition of α2ββσ. This structure is called holoenzyme (holoenzyme), remove the σ factor of the enzyme known as the core enzyme. Bacteriophage RNA polymerase is no subunit. Eukaryotic RNA polymerase sub-classified into three categories. RNA polymerase Ⅰ exist in the nucleolus, the transcription of rRNA sequence. RNA polymerase Ⅱ, the nuclear matter, the transcription of most genes, need to RNA polymerase Ⅲ present in the nuclear matter, the little
RNA polymerase transcription
several genes, such as tRNA genes, such as 5SrRNA genes. Some repetitive sequences such as Alu sequence may also be transcribed by this enzyme. The above-mentioned Its gene transcription in eukaryotes the 5 'end side, the transcription start 20 to 30 nucleotides upstream of a period between the AT-rich sequence. If the transcription start is 0 to 27 in the -33 and -27 to 21 nucleotides between nucleotides, there is a Generally is 7 nucleotides. Prokaryotes have similar The role of RNA polymerase in the Start feature is enabled ring ring (triggerloop, TL) structure in eukaryotic and prokaryotic RNA polymerase, a highly conserved region but the exact function of the transcription process is still not well understood. Stanford University, Kaplan et al found that the latest research, TL in the substrate play an important role during the selection process is the fungal toxin α Amanita Tan base (α-amanitin) the direct effect of sites. The study is published in the June 5 Previous studies showed that, TL structure directly with newly synthesized RNA 3 'end and NTP interaction, one of the conserved histidine residues directly combined with the NTP substrate. Kaplan and others replaced with other amino acids in Saccharomyces cerevisiae RNA polymerase Ⅱ TL structure His1085 residues, resulting in S. cerevisiae showed severe growth defects can not even survive. In vitro experiments showed that, His1085 able to choose the correct NTP substrate, the site mutation, the polymerase Ⅱ mixed with the wrong 2'dNTP NTP substrate and greatly increasing the possibility of substrate. Amanita Tan α can inhibit RNA polymerase Ⅱ base extension rate, reducing its substrate selectivity, but His1085 be replaced after the RNA polymerase α Amanita Tan Ⅱ on the high alkali resistance. The researchers further studied the purified RNA polymerase-α complex Amanita Tan base crystal structure of α found in Amanita Tan TL structure between the base and direct interaction. Therefore, the researchers believe, α Amanita Tan TL base through direct effect on the structure of RNA polymerase Ⅱ reduce the rate and fidelity. T7 phage substitution sequence in the transcription regulation is not replaced by σ factor cascade, nor as T4 phage enzyme modification as the core, but by the automatic features of infection in lieu of using RNA polymerase transcription in exchange for the timing regulation.
RNA polymerase
T7 is a potent E.coli phage genome is 39,936 nt, the order is known. The entire genome may have 55 genes, gene products of which 44 have been identified, and 30 are known to function in transcription can be divided into 3 groups, 30 ° C throughout the life cycle of about 25 ¢ minutes, but DNA injection is slow, the whole process for about 10 minutes, while other phage only 1 minute to complete. This is also a means of control, because no DNA into host cells can not be transcribed. 6 minutes after infection, E.coli of the RNA synthesis system systems have all been replaced by synthetic T4. In the first 6 minutes after infection, T7 phage expression RNAPol yet, so still using the host's RNA polymerase E.coli, transcription of the genes are 10 (0.3,0.4,0.5,0.6,0.65,0.7,
air force 1 shoes,1, 1.1,1.2,1.3), the 0.3 gene encoding the host restriction enzyme inhibitory protein, into the host against the degradation of the T7; 0.7 gene encodes a protein kinase, RNA polymerase can E.coli of phosphorylation, the inhibition of host RNAPol preparation; gene 1 encoding T7RNAPol polymerase, a molecular weight of 98KDa, is a single chain peptide, its recognition of the promoter is composed by the 23bp conserved sequence (-17 to 16). Late transcriptional divided into two groups (Ⅱ and Ⅲ). 6 after infection for 12 minutes after the T7 RNA polymerase transcription using their own group Ⅱ transcripts. This group contains about (1.4,1.5,1.6,1.7,1.8,2,2.5,2.8,3,3.5,3.8,
air force one low,4,5,5.7,6), which are related to the transcriptional regulation of gene 2, and its products is the E.coli polymerase inhibitors. Host RNA polymerase by the gene 0.7 product of the first phosphorylation, and then by fully
RNA polymerase inhibition
lost a role, this time I no longer needed T7 transcripts group, so the abolition of the host RNA polymerase function, but will their encoded RNA polymerase instead. Group Ⅱ T7 gene copy number and the synthesis of enzymes and structural proteins of several related, but the first group Ⅱ promoter and replication φL promoter conserved sequence in their respective bases from 2 to 7 changed Therefore, the promoter is weak, but because of its location behind the front group Ⅲ promoter, so first be transcribed into the host, rather than with the first group Ⅲ promoter competition. Another group of late gene transcription is the first group Ⅲ transcripts, which came in last, so 12 minutes after infection to express. The transcript contains about 15 genes, which encode the major head proteins, tail assembly protein and is responsible for, which makes the T7 phage particles assembled Buzhi Yu prematurely. Although this group came in last, but the promoter structure and conserved sequence is exactly the same so strong promoter, thus ensuring the final stage of structural protein synthesis. Group Ⅰ location of transcription termination of upstream neighboring gene 1.4 (between 1.3 and 1.4), this is E.coliRNA polymerase terminator) after its dissociation from T7RNA polymerase Ⅱ group were re-start transcription. Group Ⅱ and group Ⅲ use terminator Tj, as the host since the injection of slow, in the first group Ⅱ gene transcription have not yet injected group Ⅲ, still without expression. Tj termination efficiency is 90%, 10% still can extend the end of T7 transcription, Tj is located between genes 10 and 11. Major head gene 10 protein, required capacity, it ranked last in group Ⅱ was reasonable, that is strong promoter of the start, and after the termination of transcription, so that not only meets the needs of the assembly of particles, not to waste. The back row of the genes in gene 10 are encoded small amount of protein in the head and tail proteins, without a lot of transcription, extending back over the Tj can meet the needs of the intensity of transcription, which itself is a terminator of transcription regulation. The role of RNA polymerase (RNA polymerase) is the role of transcription RNA. Some RNA polymerase subunit a relatively complex structure. Such as E. coli RNA polymerase has four polypeptide chains, and another one for the synthesis of new RNA molecules σ factor, so it is the composition of α2ββσ. This structure is called holoenzyme (holoenzyme), remove the σ factor of the enzyme known as the core enzyme. Bacteriophage RNA polymerase is no subunit. Eukaryotic RNA polymerase sub-categories. RNA polymerase Ⅰ exist in the nucleolus, the transcription of rRNA sequence. RNA polymerase Ⅱ, the nuclear matter, the transcription of most genes, need to RNA polymerase Ⅲ present in the nuclear matter, the transcription of several genes, such as very few genes, such as 5SrRNA tRNA genes. Some repetitive sequences such as Alu sequence may also be transcribed by this enzyme. Above
RNA polymerase
mentioned Its transcription in eukaryotes the 5 'end side of the upstream transcription start between 20 to 30 there is a nucleotide sequence of AT-rich. If the transcription start is 0 to 27 in the -33 and -27 to 21 nucleotides between nucleotides, there is a Generally is 7 nucleotides. Prokaryotes have similar The role of RNA polymerase in the Progress in the American philosopher Ralph Waldo Emerson (RalphWaldoEmerson) said that protein linked by a prime contributor, when in fact occur in the transcription of many errors, but it also has The laboratory of the Stanford University StevenBlock study published in Nature in November above. RNA polymerase (RNAP), as long as the little people who come into contact with molecular biology the term would not have been strange, in the DNA to the expression of genetic information out of it, to become truly functional effects unit - the protein, the need for the help of RNA intermediates, but to realize this process is not the lack of this important enzyme. StevenBlock and his colleagues used an optical trap than before (opticaltrap) devices found in the more stable the process of transcription in E. coli, the enzyme about every 1,000 base there will be a mistake, but RNAP is also a good self-correction mechanisms to detect and correct these errors. In this study, StevenBlock team improved optical trap, so that magnifies 10 times
RNA polymerase
, so that researchers can directly observe the action every step of RNA polymerase, which helps depth understanding of the previous studies did not completely clear the process of gene transcription and regulation. And the use of such a device, the researchers ruled out the chance of RNAP pause and turn, can analyze the extension of a completely separate process, and they found that the RNAP in the process will move forward with a new NTP, thus lead to a slip of a RNAP base pairs to ensure the accuracy of the transcription process, which is known as Brown Jing round (brownianratchet). This research received the recognition of many scientists, University of Texas Health Center RuiSousa think this is a far the best proof of Brown Jing round, but some scientists expressed skepticism of biological DNA polymerase RNA polymerase Comparison of DNA ligase DNA ligase: DNA fragments are mainly connected between the phosphodiester bond, the connecting role in the genetic engineering work. DNA polymerase: Catalytic polymerization between the ODN. DNA fragments are mainly connected with a single deoxynucleotide phosphodiester bond between the DNA replication since to do with. DNA polymerase can only add a single nucleotide has the nucleic acid fragments of the 3 'end of the hydroxyl on the phosphodiester bond formation; and DNA ligase, two DNA fragments in the phosphodiester bond is formed between, not DNA fragments in a single nucleotide and phosphodiester bond formed between. DNA polymerase is a DNA chain as template, a single nucleotide by a phosphodiester bond formed with the template strand complementary DNA chain; and DNA ligase is the gap on the two DNA strands simultaneously connected. Therefore, no template DNA ligase. RNA polymerase (also known as the RNA replication enzyme, RNA synthetase) catalytic activity: RNA polymerase to complete the double-stranded DNA as a template, double-stranded DNA transcription structural parts removed, the transcriptional structure of double-stranded DNA remains . Eukaryotic RNA polymerase: eukaryotic transcription machinery is much more complex, there are three RNA polymerase within the nucleus: RNA polymerase I transcription of rRNA, RNA polymerase II transcription of mRNA,
nike air force one high, RNA polymerase III transcription of tRNA and other small molecular RNA. In RNA replication and transcription work. One. Templates and enzymes 1. Template RNA synthesis requires the transcription of DNA as a template, DNA double-strand chain, only an effect from the template to guide the synthesis of an RNA chain, called the template strand DNA (template strand), as opposed to the other strand chains for the coding strand (coding strand),
nike air force one, there are two asymmetric transcription meanings: First, only a portion of DNA chain segments as a transcription template (sense chain or the template strand), and second, from beginning to end is not in the same template strand single-stranded DNA on the shares. 2. RNA polymerase transcription requires RNA polymerase. By a number of prokaryotic RNA polymerase subunits: α2ββ 'as the core enzyme, only the core enzyme to transcription elongation. α2ββ'σ called holoenzyme, transcription initiation σ subunit identification is required before starting, so the whole enzyme is required for transcription initiation. Eukaryotic RNA polymerase has RNA-pol Ⅰ, Ⅱ, Ⅲ are three types of transcription of 45s-rRNA; mRNA (its precursor is the hnRNA); and the 5s-rRNA, snRNA and tRNA. 3. Template identification with the enzyme binding transcription by RNA polymerase on the template and binding sites identified. In the RNA polymerase transcription initiation before being DNA binding site called a promoter. A typical prokaryotic promoter sequences -35 and -10 region sequences TTGACA area Pribnow box is TATAAT sequence. Upstream regulatory sequences of eukaryotic transcription referred to as cis-acting element, there are TATA box,, CG box, upstream activation sequence (yeast cells), enhancer and more. Cis-acting element binding protein has a role in regulation of transcription, referred to as trans-acting factors. Trans-acting factors have been found hundreds of species, can be classified as transcription factors (TF), corresponding to RNA-pol Ⅰ, Ⅱ, Ⅲ are TF Ⅰ, TF Ⅱ, TF Ⅲ. TF Ⅱ have A, B, C, D, E, F and its various sub-categories. Basic concepts: 1. Asymmetric transcription: double meanings, one refers to the double-stranded DNA is only used as an single chain transcription template (template strand); the second is the same for different genes on a certain section of the single strand as a template chains other sections as the coding strand, the template strand is not always in the same single chain. 2. Coding strand: DNA double-stranded transcription template not used on a single chain that, in addition to its base sequence T U instead of suits, other mRNA transcripts, named after the same sequence. 3. σ (sigma) factors: Prokaryotic RNA polymerase holoenzyme composition, function is to identify the transcription initiation region, this σ factor, said σ70, in addition to different molecular weight, different functions of other σ factors. Basic Requirements: Master the difference between transcription and replication, transcription of the asymmetry, the composition of prokaryotic RNA polymerase subunit and the function of eukaryotic RNA polymerase of the classification, properties and functions of the structure of prokaryotic promoter characteristics, to understand the composition of eukaryotic RNA polymerase, transcription initiation region of the method. II. Transcription 1. Transcription initiation: the beginning of transcription is generated by RNA polymerase, template and transcription 5 'end of the first nucleotides of the initiation complex. Prokaryotes RNA5 'side is the purine nucleotide (A, G), and to retain the structure of nucleoside triphosphate, so the initiation complex is: pppG-DNA-RNA polymerase. Eukaryotic initiation complex formation before the start (PIC). For example, RNA-pol-Ⅱ transcription, is identified by a combination of various TF Ⅱ each other, and then combined with the RNA polymerase and, through TF binding to the TATA box. 2. Transcription elongation: the extension of transcription is the first nucleotide of the 3 ' -OH-based NTP added that the formation of by-phosphate bond of vinegar, so that RNA gradually from 5 'to 3' end of the growth process. In prokaryotes, because there is no separation of the cell membrane, transcription did not complete the already started translating, but also simultaneously on the same template multiple DNA transcription process. Feathery electron microscope to see tiny black spots on the graphics and feathers (polysomes), is the efficient transcription and translation of visual performance. 3. Transcription termination: termination of transcription factors in prokaryotes are divided into dependent and non-dependent Rho Rho factor categories. Rho factor has two ATP helicase enzyme activity of reconciliation, it can be combined with transcripts of the 3 'terminal region and to pause and product RNA transcribed from the DNA template. Rho factor-independent transcription termination, the RNA 3'-end products tend to form a stem-loop structure, followed by another string of oligo U. Stem-loop structure factor can no longer forward allosteric polymerase, oligomer U is not conducive to RNA dependent DNA template strand and prolapse. Therefore, no matter what kind of RNA polymerase transcription termination has stopped and the RNA product of prolapse of these two essential processes. End of eukaryotic transcription termination and processing (mRNA poly adenosine acid poly A) modified simultaneously. Last modified RNA plus points on the structure is AAAUAA sequence. Basic concepts: 1. Before the transcription initiation complex (pre-initiation complex, PIC): a eukaryotic transcription factor together with the RNA polymerase binding to DNA transcription initiation regions in front of the complexes formed. 2. Add Last Modified points: eukaryotic mRNA transcripts are not in the position of mRNA is terminated, but in hundreds of nucleotides, the study found that reading frame in the coding strand 3 'end after a set of common sequences often AATAAA, then there are quite a few GC downstream sequence, these sequences are called additive Last modified points over the modified points transcription, mRNA is cut at the modified points, then joined the polyA. 3. Rho factor: transcription termination factor prokaryotes, there is reconciliation spiral ATP enzyme activity. Or not dependent on Rho transcription termination factor. III. Eukaryotic post-transcriptional RNA processing 1. Post-transcriptional processing of eukaryotic mRNA transcripts generated by RNA, with more to be processed after the activity, a process known as post-translational modification, mRNA post-transcriptional modifications, including the first, last modification and editing. Polyadenylation and transcription termination synchronization is modified, the 5 'end of the modified cap structure mainly refers to the generation, that is 5'-pppG into 5'-pmGpppG. The process takes phosphate solution, phosphorylation and nucleotide methylation. mRNA from the hRNA processing. Eukaryotic gene intron cut off by the exon coding sequence is broken genes. Intron generally appear in the primary transcription product of hRNA. Removal of introns, the exons linked together, that is editing process. In the electron microscope to see the processing, introns are often bent sets of cords, so called lasso RNA. Now know that the editing process, the need for the various Sn-RNA and protein, and then composed of the body. And then on the body and hnRNA intron border sequences to identify with. Start with the splicing process of the enzyme with GTP to provide 3'-OH of intron 5'-end of the phosphodiester bond break for the parents to electronic attack. Exon broken 3'-OH of the intron 3'-end of the phosphodiester bond for electrophilic attack, so just break out the complete replacement of the exon intron and two exons to connected together, Therefore, this process is called the second transesterification reaction. 2. post-transcriptional processing of tRNA post-transcriptional modification of tRNA, in addition to editing process, but also includes rare tRNA chain base formation, and with the 3 'end CCA sequence. 3. rRNA post-transcriptional processing of rRNA processing in the form of multiple use of self-splicing. The formation of self-splicing of RNA itself, a special secondary structure, known as the hammerhead structure. Hammerhead structure is composed of a composite stem-loop shape, but some of the sequence must be occupied by a particular base. This RNA structure, without any protein, can hydrolyze RNA chain of a particular site on the phosphodiester bond. In other words, this is a catalytic RNA, now known as ribozymes. The discovery of ribozymes on enzymology, molecular biology, the theory of evolutionary biology is an important update, and that medicine has begun to use artificial ribozyme designed to eliminate some as RNA viruses or eliminate pathogens that are not conducive to life activities of intracellular RNA. Basic concepts: 1. Splicing modification: RNA transcripts containing the non-coding components of the primary products, by splicing to remove non-coding component, the component link encoding. The most common is the editor modified and connected by the body to assist in the second transesterification reaction, but can also be a self-editing and splicing of other splicing enzymes. 2. Exons: defined as the primary fault and its transcriptional gene product can be expressed. Or transcription product of the primary role by splicing RNA retained in the mature nucleotide sequence or gene in the mature RNA with the corresponding DNA sequences. 3. Introns: Early is defined as the non-coding DNA sequences. With the intron function has been broadening, it is recommended to use 4. And then the body: by the snRNA and protein components of the RNA protein (RNP) complexes. Its function is combined with the boundaries of intron sequences at both ends to assist in RNA splicing process. 5. Ribozyme (ribozyme): a catalytic function (enzymes) and RNA molecules. Ribozyme can play a role in the structure, contains at least three stems (RNA molecules within the pair to form a local double-stranded), 1-3, Central (RNA molecules localized single-stranded double-stranded bulge) and the consistency of at least 13 base sites. Atlas Atlas entry for more extended reading: 1
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