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Promoter region

FIGURE 6.06 Elongation of the mRNA

The beginning of RNA synthesis is shown. The DNA strands have separated at the transcription bubble. Synthesis of six bases of RNA complementary to those of the DNA template strand occurs while RNA polymerase remains at the promoter site.

Template strand \

^scription bubb

Template strand \

FIGURE 6.06 Elongation of the mRNA

The beginning of RNA synthesis is shown. The DNA strands have separated at the transcription bubble. Synthesis of six bases of RNA complementary to those of the DNA template strand occurs while RNA polymerase remains at the promoter site.

Sequence of growing RNA chain

often helpful to supply consensus promoters (or other regulatory sequences) that work well in the host organism to achieve high expression of a cloned gene from a foreign source. The use of "expression vectors" to optimize gene expression during cloning is discussed in more detail in Ch. 22.

RNA polymerase opens up the DNA to form the transcription bubble.

The core enzyme moves ahead, manufacturing RNA and leaving sigma behind at the promoter.

Manufacturing the Message

Once the sigma subunit has bound to a promoter, the RNA polymerase core enzyme opens up the DNA double helix locally to form the transcription bubble. Note that the -10 sequence, TATAAT, consists of AT base pairs and this assists in the melting of the DNA into single strands.After the DNA helix has been opened, a single strand of RNA is generated using one of the DNA strands as a template for matching up the bases. Once the RNA polymerase has bound to the DNA and initiated a new strand of RNA, the sigma subunit is no longer needed and often (though not always) detaches from the DNA, leaving behind the core enzyme. The RNA polymerase actually remains at the promoter until the new strand is eight or nine bases long. At this point, sigma leaves and the core enzyme is free to move forward and elongate the mRNA (Fig. 6.06).

The first transcribed base of the mRNA is normally an A (as in Fig. 6.06). This special A is usually flanked by two pyrimidines, most often giving the sequence CAT. Sometimes the first transcribed base is a G, but almost never a pyrimidine. Synthesis of mRNA is from 5' to 3' and proceeds at about 40 nucleotides per second. This is much slower than DNA replication (~1,000 bp/sec), but roughly equivalent to the rate of polypeptide synthesis (15 amino acids per second).

The core enzyme of RNA polymerase consists of four subunits, two a plus b and b' (Fig. 6.07). The b and b' subunits comprise the catalytic site of the enzyme. The a subunit is required partly for assembly and partly for recognizing promoters. RNA polymerase has a deep groove through the middle that can accommodate about 16 bp of DNA in the case of bacteria and about 25 bp in the case of eukaryotes such as yeast, whose RNA polymerase is larger. A thinner groove, roughly at right angles to the first, may hold the newly constructed strand of RNA.

transcription bubble Region where DNA double helix is temporarily opened up so allowing transcription to occur

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