RNA polymerases encoding

About 45% of the sequence of the RNA polymerase encoded by phage T7, which transcribes RNA from the phage DNA, is also similar to that of the Klenow fragment. Sequences of these DNA polymerases are distantly related to those of reverse transcriptases.279280 The 136-kDa polymerase y functions in mitochondria but is encoded in a nuclear gene. It is the only DNA polymerase that is inhibited by antiviral nucleotide analogs such as AZT (Box 28-C).280a b... 

Other examples include rifampin resistance due to mutations in the ipoB gene encoding the (3-subunit of RNA polymerase, or oxazolidinone resistance due to a G2576T mutation in the gene for the 23 S rRNA as central part of the 50S large ribosomal subunit. Macrolide resistance is based upon the alteration of nucleotide A2058 by a point mutation. 

The first step in creating a specific mRNA species is to construct a plasmid encoding the mRNA behind a T7, T3, or SP6 RNA polymerase promoter sequence. The S boundary of the mRNA is defined by the position of the promoter transcription start site and the S end is defined by restriction digest of the plasmid prior to in vitro transcription. 

The seqnence of amino acids in proteins is nltimately encoded in the sequence of bases in DNA. Transcription encodes this information in mRNA molecules. Each RNA polymerase transcribes a very small part of the total DNA base sequence. It follows that RNA polymerases need specific places on DNA molecules to start and to stop transcription. Getting the amino acid seqnence right is critical for protein function. It follows that getting transcription started at precisely the right place is also critical. If the mRNA base sequence is not right, the amino acid sequence in the protein will not be right. 

The next stage is to ensure that the recombinant DNA molecule is copied by the enzymes which s)mthesize nucleic acids. These DNA and RNA polymerases synthesize an exact copy of either DNA or RNA from a pre-existing molecule. In this way the DNA polymerase duplicates the chromosome before each cell division such that each daughter cell will have a complete set of genetic instructions which are then passed to the newly formed RNA by RNA polymerase. While both DNA and RNA polymerase require a preformed DNA template, some viruses (such as HIV) have an RNA genome. To duplicate that genome, and incorporate it into a bacterial or mammalian cell, the viruses encode a reverse transcriptase enzyme which produces a DNA copy from an RNA template. 

Poxvirus promoters are not recognized by eukaryotic transcription machinery. Transcription of poxviral genes is initiated only by virally encoded RNA polymerase, normally packaged alongside the DNA in the virion particles. Purified poxvirus DNA is, therefore, non-infectious. 

The Trp repressor controls the transcription of a total of five enzymes required for the biosynthesis of tryptophan (Fig. 1.22a). The genes for the five enzymes are encoded in a single operon, whereby the binding site for the Trp repressor overlaps with the promoter. The bound repressor blocks the RNA polymerase s access to the promoter, thereby inhibiting transcription. 

Some E. coli bacteriophages, including f2, MS2, R17, and Qj8, as well as some eukaryotic viruses (including influenza and Sindbis viruses, the latter associated with a form of encephalitis) have RNA genomes. The single-stranded RNA chromosomes of these viruses, which also function as mRNAs for the synthesis of viral proteins, are replicated in the host cell by an RNA-dependent RNA polymerase (RNA replicase). All RNA viruses—with the exception of retroviruses—must encode a protein with RNA-dependent RNA polymerase activity because the host cells do not possess this enzyme. 

RNA Polymerase (a) How long would it take for the E. coli RNA polymerase to synthesize the primary transcript for the E. coli genes encoding the enzymes for lactose metabolism (the 5,300 bp lac operon, considered in Chapter 28) (b) How far along the DNA would the transcription bubble formed by RNA polymerase move in 10 seconds ... 

Some strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, are resistant to rifampicin. These strains become resistant through mutations that alter the rpoB gene, which encodes the /3 subunit of the RNA polymerase. Rifampicin cannot bind to the mutant RNA polymerase and so is unable to block the initiation of transcription. DNA sequences from a large number of rifampicin-resistant M. tuberculosis strains have been found to have mutations in a specific 69 bp region of rpoB. One well-characterized strain with rifampicin resistance has a single base pair alteration in rpoB that results in a single amino acid substitution in the J3 subunit a His residue is replaced by an Asp residue. 

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