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

In the last chapter I introduced mRNA, a molecule that solves a cellular problem--namely, that the genetic instructions to make proteins reside in a membrane-enclosed nucleus while actual protein synthesis occurs outside, in the cytoplasm. mRNA molecules address this problem. They serve as temporary working copies of a genes that migrate out of the nucleus and into the cytoplasm.

Advantages of Using mRNAs

Using mRNA as an intermediary in protein synthesis offers the cell major benefits. First, 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 (in the safety of the nucleus) from genetic information use (in the bustling cytoplasm).

Using mRNAs also 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 certain protein at different times. Using intermediaries whose synthesis can be ramped up or down or turned off completely allows for this kind of control.

And third--as we'll soon see--the cell also processes mRNAs before they're used. One aspect of processing called splicing increases the number of different proteins that can be generated from one gene. This wouldn't be possible without the use of mRNAs as intermediaries. We'll get to splicing in a moment.

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 does not 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 help 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 begins.

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.

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--the strand opposite the coding strand that contains the desired codon sequence. Then, starting at the TSS, it begins moving down the DNA, building an RNA molecule that's complementary to the template strand, and therefore identical in sequence to the coding strand.

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 example 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. The 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've 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.

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 during transcription.

Second, near the end of transcription after signal poly(A) signal prompts the mRNA to be cut, a new polymerase called poly(A) polymerase enters the scene and attaches a series of about 200–250 A nucleotides to the end of the 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.

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 (including some of the upstream and downstream untranslated regions) are exons; the intervening sequences are introns.

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. Often while transcription is still underway, the pre-mRNA is acted on by a large RNA-protein complex called the spliceosome that removes introns and joins the retained exons. The final mRNA will consist of all or some of the exons connected with no intervening introns.

Splicing provides a distinct benefit to the cell: different combinations of exons can be used to produce different versions of an mRNA and thus different forms of the protein. Thus, alternative splicing enables the cell to make multiple versions of a protein from a single gene. It accomplishes this by attaching different exons together (although the exons will always be in the same order as in the pre-mRNA).

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. 

Splicing must be precise--down to the nucleotide. A one-base error at a splice junction within the protein-coding region could shift the three-letter codon reading frame and potentially scramble everything that follows.

By the time transcription and processing are complete, a usable message--a mature mRNA--has been generated from a much longer pre-mRNA. In the next chapter, we’ll discuss translation and see how the mRNA message is turned into a working protein.