8. Transcription: From DNA to RNA (1,160;9/11;pre-RL)

In the last chapter I introduced mRNA, a molecule that acts as temporary working copy of a gene that can be exported from the nucleus into the cytoplasm.

Using mRNA as an intermediary in protein synthesis offers the cell major benefits. First, as mentioned, it allows the cell to keep its valuable master copies of genes protected in the nucleus. In effect, it enables the cell to separate genetic information storage from genetic information use.

Using mRNAs has another benefit. It enables the cell to turn protein production on and off and to control protein levels. With a few exceptions, every cell has the same genome. But different cell types express different genes. In addition, a cell might require more or less of a protein at different times. Using intermediaries whose synthesis can be ramped up or down or turned off completely enables this kind of control.

The arrangement I've described thus far resembles a protected archive that never lends out its originals. Instead, it produces disposable working copies--copies that can be made by the hundreds, sent out where they are needed, and destroyed when their usefulness has ended.

A third benefit of using mRNA as an intermediary--as we'll soon see--is that the cell can process mRNAs before they're used. One aspect of processing is splicing, or editing. Splicing an mRNA can increase the number of different proteins that can be generated from a single gene. We'll take a look at splicing later in the chapter.

Identifying Genes

Up to this point, we’ve treated the genome as if it was one long, continuous message. In fact, only certain stretches--genes--are used to make proteins. Roughly 98.5% of the genome doesn't code for proteins. Some of this noncoding DNA has well-understood roles--for example, regulating the use of specific genes--but the function, if any, of much of it is still being worked out.

So how does the cell identify the genes amid long stretches of DNA nucleotides? 

Among the most important clues are DNA sequences called promoters that are located just in front, or upstream, of genes. Promoters position the transcription machinery--the proteins that will be involved in transcription--at the transcription start site (TSS), which is the exact nucleotide at which mRNA synthesis will begin.

Transcription is performed by the enzyme RNA polymerase II, which, as mentioned, attaches to the promoter sequence prior to transcription. RNA polymerase II does not work alone, however. As many as fifty or sixty additional proteins may help assemble and operate the machinery that initiates transcription, with still others regulating the process.

No overseer directs these molecules to their proper positions. Each protein responds only to nearby molecular shapes, electrical charges, and chemical attractions. Yet, together, they assemble a machine capable of locating the beginning of a gene and copying it with remarkable precision.

The Transcription Process

Once the transcription machinery has assembled, it opens a small bubble in the double-stranded DNA, giving RNA polymerase II access to the template strand that's opposite the coding strand. Then, starting at the TSS, it moves down the DNA, building an RNA molecule that's complementary to the template strand, and therefore identical in sequence to the coding strand (except that "U" replaces "T").

For example, if the first DNA nucleotide on the template strand is "G," RNA polymerase II will match it with a complementary "C" RNA nucleotide. If the next DNA nucleotide is "T," the enzyme will match it with an "A" RNA nucleotide and then chemically link the two RNA nucleotides--the "C" and the "A." These will be the first two RNA nucleotides in our mRNA.

This process continues. RNA polymerase II progresses down the template strand extending the length of the mRNA. The enzyme typically travels along a human gene at roughly 50 nucleotides per second, although it frequently pauses. As the length of the mRNA increases, it gradually peels away and the double-stranded DNA comes back together behind it.

Near the end of the mRNA transcript, a sequence in the gene called the poly(A) signal (AAUAAA) prompts processing enzymes to cut and release the mRNA about 10-30 nucleotides further on. RNA polymerase continues some distance further before detaching from the DNA.

mRNA Processing

Up to now, we’ve treated the mRNA as if it were ready to use once it's transcribed. It isn’t. The mRNA first generated by RNA polymerase--the one we just created--is called a pre-mRNA. It must be processed to create the final mature mRNA--the RNA that will be used to make a protein. Processing occurs in the nucleus and involves three steps, two of which happen while the mRNA is still being transcribed.

First, the cell adds a 5′ cap--a modified G (guanine) nucleotide--to the front end of the mRNA. This cap protects the mRNA from degradation and helps recruit proteins involved in processing, export, and translation. Capping occurs soon after transcription starts, while the RNA is still being transcribed.

Second, after transcription--that is, after the poly(A) signal prompts the mRNA to be cut free--a new polymerase called poly(A) polymerase attaches a series of about 200–250 A nucleotides to the end of the mRNA transcript. This poly(A) tail stabilizes the molecule and helps it exit the nucleus. Over time, enzymes will gradually shorten the poly(A) tail. Once it becomes sufficiently short, the mRNA usually becomes unstable and is degraded. In this sense, mRNAs are born with something resembling a molecular lifetime.

Third, and finally, there is mRNA splicing, or editing. This is where the cell gets very tricky! Within the transcribed region of a gene are sequences that don't actually code for the protein. The coding portions of a gene are exons. The intervening sequences are introns. Splicing often begins before the pre-mRNA is completed. Let's see how this works.

mRNA Splicing

The mRNA that's produced by the RNA polymerase II--the pre-mRNA--contains all the gene's exons and all its introns. Usually while transcription is still underway, the pre-mRNA is acted on by multiple large RNA-protein complexes called spliceosomes that remove introns and join the retained exons. The final mRNA will consist of all or some of the exons connected with no intervening introns.

Alternative splicing allows different combinations of exons to be used to produce different versions of an mRNA--and thus different forms of the protein--from a single gene.

For example, imagine a gene with a transcribed region that consists of six exons separated by five introns. In one case, a protein might be produced by splicing together every exon (exons 1-6). Another protein with a slightly different role might be made by combining only exons 1, 3, 5, and 6. Another might be comprised of exons 2, 3, 4, and 5. Most human genes contain introns, and many can be spliced in different ways to produce protein variants. 

Thus, a gene is not always a rigid construction for one and only one protein. It can be more like a collection of useable segments from which the cell constructs different--but carefully specified--messages.

Realize that a spliceosome must identify exact boundaries of an intron within a molecule that may contain tens of thousands of nucleotides. It must cut at the correct positions, bring together two exons that were previously far apart, and join them without losing or adding even a single nucleotide.

Being off by one nucleotide would alter every codon that follows. In other words, if the reading of triplet codons shifts by one or two nucleotides, then all of the subsequent three-letter codons will be out of step (i.e., in the wrong frame) and thus nonsensical.

To recap, the cell does not wait for RNA polymerase to finish before beginning to process the RNA. While the back end of the message is still being written, proteins are already modifying its front end and spliceosomes may be removing introns. The molecule is edited while it is still under construction.

By the end of this process, the cell has located one useful stretch within billions of DNA letters, copied its information into another kind of molecule, removed specific unwanted portions, joined the retained portions with nucleotide-level precision, and prepared the finished message for export--all without altering the original gene. The mature mRNA is now ready to carry that information into the cytoplasm, where it will direct the construction of a protein.