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RNA
Replication
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Replicase |
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RNA
Replication
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Replicase -
Replication of phage RNA takes places by viral RNA replicase (RNA-dependent RNA polymerase).
As mentioned previously, the enzyme consists of four subunits. Three (and α, γ, δ) are host polypeptides, while one (/3 subunit) is a virus specified polypeptide.
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The γ and the δ subunits are the protein synthesis elongation factors EF- Tu and EF- Ts, respectively.
The α subunit is a component of the 305 ribosomal subunit. For plus strand replication a host specific factor, HFI, is required.
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The replacases of the different phages show high specificity/ and recognize only the RNAs of phages of the same group. Their recognition site is a proper steric arrangement of two CCC sequences.
One of these is present at the 5' end of all phage RNAs, while the other is present at slightly different distances in the different groups of viruses.
The secondary structure of the RNA fixes the relative positions of the c two, CCC sequences.
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Replication of phage RN A has two unique features. Firstly, RNA serves as a template for the synthesis of an RNA str and (RNA -+ RNA). Secondly, there is direct generation of single strands without the formation of duplex structures. Both the template and the product arc single stranded.
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Replication of RNA takes place ill two steps. In the first step the plus strand is used as a template for the synthesis of a complementary single stranded minus strand.
For plus strand replication the enzyme RN A replicase and a host-specific factor (BFI -or host factor) are required.
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The requirement of another host specific factor, HFII, has been suggested, but it appears that this factor IS necessary only when the template is contaminated by an inhibitor - In the second step the minus strand is used as a template to produce the viral plus strand,.
Minus strand replication can be
brought about by the β, γ, δ subunits of RNA replicase, and no host factor is required.
The minus strand functions only as a template for positive strand synthesis. The plus strand has multiple functions:
(i) It acts a template for minus strand synthesis,
(ii) it acts in protein synthesis and
(iii) it forms the genome which is packaged into phage
particles.
Two types of RNA replication intermediates have been found in, bacterial cells infected with RNA labelled virions. They are the replicative form (RF), and the replicative intermediate (RI).
The replication form is entirely double-stranded, and is completely resistant to single-strand RNase. It is produced by the synthesis or a complementary minus strand on the infective viral positive strand.
It therefore consists
of one positive and one negative strand. Subsequent synthesis of a third progeny positive strand on the RF converts it into the RI.
The replicative intermediate has a double stranded backbone consisting of one positive and one/negative strand, with one or two single stranded fails. Is partially resistant to RNase.
The single-Stranded tails are the progeny strands which are' finally released from the replicative intermediate.
The replicative intermediates show two possible types of replication, semiconservative and conservative. In semiconservative replication the newly formed ,strand (Wavy line) is hydrogen bonded to the parent strand. As synthesis proceed, it displaces the pre-existing positive strands, (thick line.
The result or semiconservative replication is two positive strands, one of which is the original parental strand and the other the newly synthesized progeny strand. In conservative replication the duplex, of plus and minus strand remains intact, except in the region of the growing point where synthesis of new strands takes place.
In conservative replication all the strands
formed are positive progeny strands. In semiconservative replication the parental strand is exposed to RNase, attack during replication, while in the conservative replication it is not.
In the experiments described above, the RNA extracted from infected cells was found to be in the form of duplex molecules which were resistant to single strand -RNase. It was, however, suggested at duplex formation was the result of removal of protein during the extraction process.
This has led to a model/for RNA replication in which the duplex region is restricted to the RNA sequences covered by the enzyme. Both template and product remain in the Bingle stranded condition. With the extraction of protein, they form a duplex structure. In viyo duplex formation is prevented by immediate folding of the strands and the acquisition of secondary structure of b
In phage RNA the adenylic acid residue is added to the 3' end of both strands after replication. Viral RNA has a -CCA-3' terminal as-in-tRNA. The synthesis of viral replicase subunit begins at the end of the template. The terminal A is, however, passed/over.
The first nuc1eotidC-of the newly synthesized strand is a G nucleotide which, is laid opposite to the C (last-but-one nucleotide). On completion of the strand the 5'-terminal nucleotide, pppG, directs the addition of a C, and the enzyme finally adds a final A to the product. That the terminal-CCA sequence is restored.
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