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Modern Genetic Analysis Chapter 3. Gene Function
Making Functional Transcripts

Exactly how is a gene coding sequence, which is a relatively small segment of DNA embedded in a much longer DNA molecule (the chromosome), transcribed into a single-stranded RNA molecule of correct length and nucleotide sequence? In considering the several distinct steps involved, we will first examine the nature of the template.

Transcription Uses One DNA Strand as Template

Transcription relies on the complementary pairing of bases. The two strands of the DNA double helix separate locally, and one of the separated strands acts as a template (alignment guide) for RNA synthesis. In the chromosome overall, both DNA strands are used as templates, but in any one gene only one strand is used, and in that gene it is always the same strand (Figure 3-2).

Next, free ribonucleotides that have been synthesized elsewhere in the cell align on the DNA template, "called in" by the their complementary bases in the template. The free ribonucleotide A aligns with T in the DNA, G with C, C with G, and U with A. The process is catalyzed by the enzyme RNA polymerase, which attaches and moves along the DNA adding ribonucleotides to the growing RNA as shown in Figure 3-3a. Hence, already we see the two principles of base complementarity and binding proteins (in this case the RNA polymerase) in action.

RNA growth is always in the 5 3 direction; in other words, nucleotides are always added at a 3 growing tip, as shown in Figure 3-3b. Because of the antiparallel nature of nucleotide pairing, the fact that RNA is synthesized 5 3 means that the template strand must be oriented 3 5. As RNA polymerase molecules move along the gene, the growing RNA molecules can be seen under the electron microscope, as shown in Figure 3-4.

Base complementarity also means that the nucleotide sequence in RNA is the same as in the nontemplate strand, except that the T's are replaced by U's, as shown in Figure 3-5 on page 56. The nontemplate strand matches the RNA transcript in direction and base sequence (thus facilitating comparisons). When DNA base sequences are cited, by convention it is the sequence of the nontemplate strand that is given, even though RNA is transcribed on the template strand. This distinction is extremely important to keep in mind during discussions of transcription.

MESSAGE
Transcription is asymmetric: only one strand of the DNA of a gene is used as a template for transcription. This strand is in 3 5 orientation, and RNA is synthesized in the 5 3 direction.

RNA Polymerases

In most prokaryotes, a single RNA polymerase does the job of transcribing all types of RNA. However, eukaryotes have three different RNA polymerases, which specialize as follows:

1. RNA polymerase I (Pol I) transcribes rRNA genes.
2. RNA polymerase II (Pol II) transcribes protein-coding genes.
3. RNA polymerase III (Pol III) transcribes other functional RNA genes (for example, tRNA genes).

In eukaryotes transcription of nuclear chromosomes takes place entirely within the nucleus, and the transcripts then move through nuclear pores out into the cytosol (the liquid phase of the cytoplasm), where translation occurs. Organelle genomes are transcribed within the organelle, and translation is also within the organelle. Since prokaryotes have no nucleus, there is no comparable movement of transcripts, and translation can take place immediately, right on the growing transcript.

Now that we've met the players involved, let's look at the three distinct stages of transcription: initiation, elongation, and termination, using the system of the gut bacterium E. coli as an example.

Initiation

A DNA sequence to which RNA polymerase binds to initiate transcription is termed a promoter. A promoter is part of the regulatory region adjacent to the coding region of a gene. Remember, since an RNA transcript is made in the 5 3 direction, the convention is to view the gene in the 5 3 orientation, too (the orientation of the nontemplate strand), even though transcription actually starts at the 3 end of the template strand. By convention the first-transcribed end of the gene is called the 5 end. In other words, the gene is labeled in the same way as its RNA transcript. Using this view, the promoter is at the beginning of the gene and, so, is said to be at the 5 end of the gene, and the regulatory region is called the 5 regulatory region (Figure 3-6).

Figure 3-7 shows the promoter sequences of 13 different transcription initiation points on the E. coli genome. The diagram shows the base sequences that appear just before the first-transcribed base, designated the "initiation site." Two regions of partial similarity appear in virtually each case. These regions have been termed the 35 (minus 35) and 10 regions because of their locations relative to the transcription initiation point. At the bottom of Figure 3-7 the promoter consensus sequence is given, in other words the "average" promoter sequence. RNA polymerase scans the DNA for a promoter sequence, binds to the DNA at that point, then unwinds it and begins the synthesis of an RNA molecule at the transcriptional initiation site. Hence, we see that the principle of DNA binding is a result of interactions between the protein (here, the RNA polymerase) and a specific base sequence in the DNA.

Elongation

The RNA polymerase moves along the DNA, maintaining a transcription "bubble" to expose the template strand, and catalyzes the 3 elongation of the RNA strand. The polymerase compares free ribonucleotide triphosphates with the next exposed base on the DNA template and, if there is a complementary match, adds it to the chain. The energy for the reaction is derived from splitting the high-energy triphosphate into the monophosphate and releasing the inorganic diphosphates, according to the following general formula:

Termination

When RNA polymerase recognizes specific nucleotide sequences in the DNA that act as signals for chain termination, the RNA strand and the polymerase are released from the DNA template. E. coli has two main mechanisms for termination; we will look only at the direct termination mechanism here. The terminator sequences consist of about 40 bp, ending in a GC-rich stretch that is followed by a run of six or more A's on the template strand. The corresponding GC sequences on the RNA are arranged in such a way that the transcript in this region is able to form complementary bonds with itself, as can be seen in Figure 3-8. The resulting double-stranded RNA section is called a hairpin loop. It is followed by the terminal run of U's that correspond to the A residues on the DNA template. The hairpin loop and section of U residues appear to serve as a signal for the release of RNA polymerase and termination of transcription.

RNA Processing in Eukaryotes

Transcription works in much the same way in eukaryotes as in prokaryotes; that is, there are specific promoter sequences to which the RNA polymerase binds, and the polymerase moves along the gene synthesizing RNA in the 5 3 direction. However, in eukaryotes the initial product of transcription, the primary RNA transcript, is processed in several ways before its transport to the cytosol. These processing steps are all performed by specific proteins that bind to the RNA. Figure 3-9 depicts some events in the processing of the primary transcript of a protein-coding gene. Until it reaches its final, mature form, the primary transcript is sometimes called pre-mRNA. First, during transcription, a cap consisting of a 7-methylguanosine residue is added to the 5 end of the transcript, linked by a triphosphate bond. Then an AAUAAA sequence near the 3 end is recognized by an enzyme that cuts off the end of the RNA approximately 20 bases farther down. At this time a stretch of 150 to 200 adenine nucleotides called a poly(A) tail is added at the cut 3 end. Next, a crucial splicing step removes any introns from the RNA transcript, converting pre-mRNA into mature mRNA. Splicing is discussed next.

Split genes.

For many eukaryotic genes the capped and tailed transcripts are shortened by the elimination of internal introns before transport into the cytoplasm. In most higher eukaryotes studied, this is found to be true not only for mRNA but also for rRNA, and even for tRNA in some cases.

Figure 3-10a shows the general structure of a eukaryotic gene with some of the main landmarks relating to transcription, translation, and intron organization. Figure 3-10b diagrams the processing of the primary transcript, including intron excision. The pre-mRNA is processed by a series of "splicing" reactions, much in the same way that a tape-recorded message can be cut and pasted back together. Splicing removes the introns and brings together the coding regions, the exons, to form an mRNA, which now consists of a sequence that is completely colinear with the protein.

Mechanism of exon splicing.

Exon-intron junctions of mRNAs show specific sequences that are highly conserved; in other words, they are the same in most introns in most species. These sequences are functional in the splicing reactions. For example, there is a GU at the 5 splice site of the intron and an AG at the 3 splice site in virtually all cases examined ("the GU-AG rule"; see Figure 3-11). Other less well conserved sequences are found flanking these. These common configurations of the pre-mRNA are recognized by small nuclear ribonucleoprotein particles, or snRNPs, which catalyze the cutting and splicing reactions. The snRNPs are complexes of protein and small nuclear RNA (snRNA). Through base-pair complementarity of their snRNAs, the snRNPs may help to align the splice sites by hydrogen bonding to the sequences at the exonintron boundaries (Figure 3-12). Next the intron is excised. A lariat-shaped intermediate is produced and then released to yield the final mRNA. Figure 3-13 portrays the lariat model. Here the 5 splice site is first cleaved [Figure 3-13(a)] in a reaction that creates a loop [Figure 3-13(b)]. The newly formed 3 OH group then cleaves the 3 splice site [Figure 3-13(b)], releasing the partly looped intron and at the same time joining the two exons [Figure 3-13(c)]. The formation of the lariat also requires a conserved sequence in the intron, and attachment is always at a highly conserved adenine base.

During the process of splicing, the snRNPs, the primary transcript, and associated factors all come together to form a high-molecular-weight (60S) ribonucleoprotein complex, called a spliceosome (Figure 3-14), which catalyzes the splicing reactions just described.

One exceptional case of RNA splicing occurs in the ciliate protozoan Tetrahymena, in which the splicing reaction is catalyzed by the transcribed RNA molecule itself. This extraordinary finding was the first demonstration that an RNA molecule in some cases can act as an enzyme and catalyze a specific biological reaction. These RNAs with enzymatic activities have been termed ribozymes.

MESSAGE
Eukaryote mRNA is processed by 5 capping, 3 polyadenylation, and intron excision.

Once a primary transcript has been fully processed into a mature mRNA molecule, translation into protein can take place. Before considering the mechanism of translation, we must understand the nature of protein structure.

© 1999 by W. H. Freeman and Company. All rights reserved.