"Gene expression" refers to the series of processes that lead from the molecular genotype (the DNA base pair sequence of the gene) to the molecular phenotype (the protein coded for by the gene). Both transcription and translation have an amplifier effect. For example, if transcription from some gene happens 20 times (in some time interval), and then each of the 20 mRNA molecules gets translated 50 times, the overall result is the synthesis of 1,000 copies of the protein coded for by the gene. Today we will look at getting the mRNA synthesized, ready to go to the cytoplasm to get translated.


1. What is the basic enzymology of RNA synthesis; i.e., making the primary transcript?

Figure 10.6 shows the basic polymerization reaction catalyzed by RNA polymerases. With the template DNA strand getting read in the 3'-5' direction, the RNA gets synthesized 5'-3', using nucleoside triphosphates that get incorporated (as monophosphates) into the growing RNA chain.

Figure 10.7 shows the structure of a bacterial RNA polymerase, a large protein consisting of six distinct polypeptide chains. Eukaryotic RNA polymerases are even larger, and moreover there are three types, each responsible for making different classes of transcripts (described on page 406). The one we will consider most is RNA polymerase II, which synthesizes all the primary transcripts that eventually become mRNAs. The other two RNA polymerases, I and III, synthesize the rRNAs and tRNAs.


2. What makes transcription start and stop at the correct places, to result in RNA copies of complete genes, rather than copies of fragments of genes?

For prokaryotes, Figure 10.8 shows eight "promoters", short (20 to 200 bp) regions in the DNA to which RNA polymerase can bind, determining the start sites and direction of transcription. The actual promoter sequences shown in the figure are from E. coli. What is shown, for each promoter sequence, is one strand, 5'-3', of the DNA just "upstream" from the transcription start site, which is labelled "+1" in the figure. The actual mechanism by which RNA polymerase "opens up" the double helical DNA has just been solved recently, as summarized in this figure from the journal Science in 2004.

In eukaryotes, the transcription start sequences are somewhat different, but the principle is similar. Figure 11.25 (page 471) shows a representation of how transcription starts at the beginning of a typical eukaryotic gene. Note that the transcription initiation process involves additional proteins (beyond just RNA Polymerase) and an additional DNA region (beyond just the promoter).

For termination of transcription, Figure 10.9 shows one mechanism. There is an inverted repeat sequence in the DNA, and as transcription passes this point, the newly made region of RNA folds into a "hairpin" structure. This hairpin structure somehow interacts with the RNA polymerase enzyme, causing the enzyme to fall off the DNA.
The example shown in this figure is from E. coli. In eukaryotes, the details may be somewhat different (and varied), but the principle is similar, involving DNA sequences that cause RNA polymerase to release from the DNA.


3. In eukaryotes, what are the processing steps, in the nucleus, involved in converting a primary transcript into an mRNA?

There are enzyme complexes in the nucleus that carry out the modifications shown in Figure 10.12.

A "cap", methylated guanosine, is added (by a 5'-5' bond) to the 5'end.

A "tail" of 50 to 200 or so adenosines is added to the 3' end.

The process called "splicing" removes all of the introns of the primary transcript and joins the exons together. Some details of how this occurs are shown in Figure 10.13. {Figure 10.14 presents more elaborate details (for which you are NOT responsible), showing the complex molecular interactions involved.} The splicing process is usually going on even while the primary transcript is still being made. Thus, in these cases the primary transcript never really exists as a full length molecule.

So, all of this is going on in the nuclei of your cells, resulting in mRNAs that will then be used to synthesize proteins in the cytoplasm.