Genes VII

Transcription takes place by the usual process of complementary base pairing, catalyzed and scrutinized by the enzyme RNA polymerase. Figure 9.3 illustrates the general nature of transcription. RNA synthesis takes place within a "transcription bubble," in which DNA is transiently separated into its single strands, and one strand is used as a template for synthesis of the RNA strand. As RNA polymerase moves along the DNA, the bubble moves with it, and the RNA chain grows longer.

The RNA chain is synthesized from the 5′ end toward the 3′ end. The 3′ VOH group of the last nucleotide added to the chain reacts with an incoming nucleoside 5′ triphosphate. The incoming nucleotide loses its terminal two phosphate groups (γ and β); its α group is used in the phosphodiester bond linking it to the chain. The overall reaction rate is ~40 nucleotides/second at 37 XC (for the bacterial RNA polymerase); this is about the same as the rate of translation (15 amino acids/sec), but much slower than the rate of DNA replication (800 bp/sec).

The structure of the bubble within RNA polymerase is shown in the expanded view of Figure 9.4. As RNA polymerase moves along the DNA template, it unwinds the duplex at the front of the bubble (the unwinding point), and rewinds the DNA at the back (the rewinding point). The length of the transcription bubble varies with the phase of the elongation reaction from 12 V20 bp (see later), but the length of the RNA-DNA hybrid region within it is shorter (for review see 68).

There is a major change in the topology of DNA extending over ~1 turn, but it is not clear how much of this region is actually base paired with RNA at any given moment. Certainly the RNA-DNA hybrid is short and transient. As the enzyme moves on, the DNA duplex reforms, and the RNA is displaced as a free polynucleotide chain. About the last 25 ribonucleotides added to a growing chain are complexed with DNA and/or enzyme at any moment.

What is the length of the RNA-DNA hybrid in the transcription bubble? The classic view of transcription was that the hybrid region extends for ~12 bp, as suggested by indirect evidence about the structure of the RNA region within RNA polymerase. Attempts to make more direct measurements typically use a situation in which an elongating RNA polymerase is halted at a particular position by withholding the nucleotide that is required to add the next base to the RNA chain. Then the reactivity of bases in RNA or DNA at particular positions is tested. The results vary in suggesting a length for the RNA-DNA hybrid region between 3 and 9 base pairs. Of course, the answer may be different for different RNA polymerases.

Bacterial RNA polymerase has overall dimensions of ~90 95 160 Å. Yeast RNA polymerase is larger but less elongated. Structural analysis shows that they share a common type of structure, in which there is a "channel" or groove on the surface ~25 Å wide that could be the path for DNA. This is illustrated in Figure 9.5 for the example of yeast RNA polymerase. The length of the groove could hold 16 bp in the bacterial enzyme, and ~25 bp in the yeast enzyme, but this represents only part of the total length of DNA bound during transcription. Aside from this general description, we cannot yet relate the features of the enzyme to the generalized structure shown in Figure 9.4.

RNA polymerase controls the entry of incoming nucleotides. Probably the enzyme allows phosphodiester bond formation to proceed only when a complementary nucleotide matches the template base. The nucleotide is expelled if it does not fit; then another can enter. The mechanism for discrimination is not known, but probably does not rely upon base pairing directly, because some base analogs that cannot pair are incorporated well.

The traditional view of elongation has been that it is a monotonic process, in which the enzyme moves forward 1 bp along DNA for every nucleotide added to the RNA chain. Changes in this pattern occur in certain circumstances, in particular when RNA polymerase pauses. One type of pattern is for the "front end" of the enzyme to remain stationary while the "back end" continues to move, thus compressing the footprint on DNA. After movement of several base pairs, the "front end" is released, restoring a footprint of full length. This gave rise to the "inchworm" model of transcription, in which the enzyme proceeds discontinuously, alternatively compressing and releasing the footprint on DNA. However, it may be the case that these events describe an aberrant situation rather than normal transcription (502, 504).

We now have a direct view of the active site from a crystal structure of a phage T7 RNA polymerase engaged in transcription (925). The RNA-DNA hybrid region is only 3 base pairs long. It is compressed in the active site; the conformation of the enzyme remains essentially the same while several nucleotides are added, and the transcribed template strand is "scrunched" in the active site. The active site can hold a transcript of 6-9 nucleotides.

One means of producing a pause is to arrest elongation (typically by omission of precursor nucleotides during an in vitro reaction). When the missing nucleotide is restored, the enzyme can overcome the block by cleaving the 3′ end of the RNA, to create a new 3′ terminus for chain elongation. The cleavage involves accessory factors in addition to the enzyme itself. In the case of E. coli RNA polymerase, the proteins GreA and GreB release the RNA polymerase from elongation arrest. Animal cell RNA polymerase requires the accessory factor (TFIIS), which enables the polymerase to cleave a few ribonucleotides from the 3′ terminus of the RNA product.

The catalytic site of RNA polymerase undertakes the actual cleavage in each case. There have been differences of opinion concerning the change in the enzyme that occurs at this time. One view is that there is an internal reorganization of structure, in which the catalytic center moves relative to the rest of the enzyme. The alternative model shown in Figure 9.6 suggests that the enzyme as a whole "backtracks" on the DNA. The 3′ terminus of the RNA is exposed in single-stranded form, and the RNA-DNA hybrid region reverses its position. Cleavage restores a normal elongation complex. This model is supported by more recent measurements showing a constant distance between the catalytic center and the "front end" (505).

We see therefore that RNA polymerase has the facility to unwind and rewind DNA, to hold the separated strands of DNA and the RNA product, to catalyze the addition of ribonucleotides to the growing RNA chain, and to adjust to difficulties in progressing by cleaving the RNA product and restarting RNA synthesis (with the assistance of some accessory factors).

This section updated 1-10-2000