In 1970, Oscar Miller, Jr., Barbara Hamkalo, and Charles Thomas used electron microscopy to examine cellular contents and demonstrate that RNA is transcribed from a DNA template. They saw Christmas-tree-like structures within the cell: thin central fibers (the trunk of the tree) to which were attached strings (the branches) with granules (Figure 10.3a). The addition of deoxyribonuclease (an enzyme that degrades DNA) caused the central fibers to disappear, indicating that the “tree trunks” were DNA molecules. Ribonuclease (an enzyme that degrades RNA) removed the granular strings, indicating that the branches were RNA. Their conclusion was that each “Christmas tree” represented a gene undergoing transcription (Figure 10.3b). The transcription of each gene begins at the top of the tree; there, little of the DNA has been transcribed, and the RNA branches are short. As the transcription apparatus proceeds down the tree, transcribing more of the template, the RNA molecules lengthen, producing the long branches at the bottom.

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THE TRANSCRIBED STRAND The template for RNA synthesis, as for DNA synthesis, is a single strand of the DNA double helix. Unlike replication, however, the transcription of a gene takes place on only one of the two nucleotide strands of DNA (Figure 10.4). The nucleotide strand used for transcription is termed the template strand. The other strand, called the nontemplate strand, is not ordinarily transcribed. Thus, within a gene, only one of the nucleotide strands is normally transcribed into RNA (there are some exceptions to this rule). Although only one strand within a single gene is normally transcribed, different genes may be transcribed from different strands, as illustrated in Figure 10.5.

During transcription, an RNA molecule that is complementary and antiparallel to the DNA template strand is synthesized (see Figure 10.4). The RNA transcript has the same polarity and base sequence as the nontemplate strand, except that it contains U rather than T. TRY PROBLEM 16

THE TRANSCRIPTION UNIT A transcription unit is a stretch of DNA that encodes an RNA molecule and the sequences necessary for its transcription. How does the complex of enzymes and proteins that performs transcription—the transcription apparatus—recognize a transcription unit? How does it know which DNA strand to read and where to start and stop? This information is encoded by the DNA sequence.

Included within a transcription unit are three critical regions: a promoter, an RNA-coding sequence, and a terminator (Figure 10.6). The promoter is a DNA sequence that the transcription apparatus recognizes and binds. It indicates which of the two DNA strands is to be read as the template and the direction of transcription. The promoter also determines the transcription start site, the first nucleotide that will be transcribed into RNA. In many transcription units, the promoter is located next to the transcription start site but is not itself transcribed.

The second critical region of the transcription unit is the RNA-coding region, a sequence of DNA nucleotides that is copied into an RNA molecule. The third component of the transcription unit is the terminator, a sequence of nucleotides that signals where transcription is to end. Terminators are usually part of the RNA-coding sequence; that is, transcription stops only after the terminator has been copied into RNA.

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Molecular biologists often use the terms upstream and downstream to refer to the direction of transcription and the locations of nucleotide sequences surrounding the RNA-coding sequence. The transcription apparatus is said to move downstream as transcription takes place: it binds to the promoter (which is usually upstream of the start site) and moves toward the terminator (which is downstream of the start site).

When DNA sequences are written out, often the sequence of only one of the two strands is listed. Molecular biologists typically write the sequence of the nontemplate strand because it will be the same as the sequence of the RNA transcribed from the template strand (with the exception that U in RNA replaces T in DNA). By convention, the sequence on the nontemplate strand is written with the 5′ end on the left and the 3′ end on the right. The first nucleotide transcribed (the transcription start site) is numbered +1; nucleotides downstream of the start site are assigned positive numbers, and nucleotides upstream of the start site are assigned negative numbers. So, nucleotide +34 would be 34 nucleotides downstream of the start site, whereas nucleotide −75 would be 75 nucleotides upstream of the start site. There is no nucleotide numbered 0.