Transcription

Transcription differs between prokaryotes (single-celled organisms that lack membrane-bound nuclei and membrane-enclosed organelles) and eukaryotes (organized around histone proteins), but the fundamentals are the same. Transcription of a gene begins when the enzyme RNA polymerase binds to DNA at the promoter region of that gene. A promoter is a DNA sequence onto which the transcription machinery, including RNA polymerase, binds and initiates transcription. Promoters exist a certain distance upstream of the genes they regulate.

Transcription requires the DNA double helix to partially unwind in the region of messenger RNA (mRNA) synthesis. The region of unwinding is called a transcription bubble. Transcription always proceeds from the same DNA strand for each gene, which is called the template strand. The mRNA product is complementary and antiparallel to the template strand and is almost identical to the other DNA strand, called the coding strand. There are two differences: 1) in mRNA, all of the T nucleotides are replaced with U nucleotides; 2) in mRNA, the sugar phosphate “backbone” of the molecule is ribose, whereas for DNA, the sugar is deoxyribose. In an mRNA-DNA strand pairing during transcription, A can bind U via two hydrogen bonds, just as in A–T pairing in a DNA double helix. Messenger RNA is always synthesized in the 5′ to 3′ direction.

Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals. One is protein-based and the other is RNA-based. Rho-dependent termination is controlled by the rho protein, which tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. The interaction with rho releases the mRNA from the transcription bubble.

Unlike prokaryotes, eukaryotes have multiple, linear chromosomes. Eukaryotic mRNAs typically specify a single protein, as opposed to the mRNA transcribed from multi-gene units called operons. The structure of a generalized eukaryotic protein-coding gene and its product is shown below.

Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few key differences. The most important difference between prokaryote and eukaryote transcription is due to the latter’s membrane-bound nucleus and organelles. With the genes bound in a nucleus, the eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its mRNA from degrading before it is translated. Eukaryotes also employ three different polymerases that each transcribe a different subset of genes.

RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs. Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation. For clarity, we will only use the term “mRNAs” to describe only the mature, processed molecules that are ready to be translated. RNA polymerase II is responsible for transcribing the overwhelming majority of eukaryotic genes.

RNA Polymerase II Promoters and Transcription Factors

Eukaryotic promoters are much larger and more intricate than prokaryotic promoters. However, both have a sequence similar to the -10 sequence of prokaryotes. In eukaryotes, this sequence is called the TATA box, and has the consensus sequence TATAAA on the coding strand. It is located at -25 to -35 bases relative to the initiation (+1) site.

Like prokaryotic cells, the transcription of genes in eukaryotes requires the action of an RNA polymerase to bind to a DNA sequence upstream of a gene in order to initiate transcription of that gene. However, unlike prokaryotic cells, the eukaryotic RNA polymerase requires other proteins, or transcription factors, to facilitate transcription initiation. RNA polymerase by itself cannot initiate transcription in eukaryotic cells.

Eukaryotic Elongation and Termination

Following the formation of the pre-initiation complex, the polymerase is released from the promoter, and elongation is allowed to proceed as it does in prokaryotes, with the polymerase synthesizing pre-mRNA in the 5′ to 3′ direction. As discussed previously, RNA polymerase II transcribes the major share of eukaryotic genes, so in this section we will focus on how this polymerase accomplishes elongation and termination.

Although the enzymatic process of elongation is essentially the same in eukaryotes and prokaryotes, the DNA template is considerably more complex. When eukaryotic cells are not dividing, their genes exist as a diffuse mass of DNA and proteins called chromatin. The DNA is tightly packaged around charged histone proteins at repeated intervals. These DNA–histone complexes, collectively called nucleosomes, are regularly spaced and include 146 nucleotides of DNA wound around eight histones like thread around a spool. For polynucleotide synthesis to occur, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome. This is accomplished by a special protein complex called FACT, which stands for “facilitates chromatin transcription.” This complex pulls histones away from the DNA template as the polymerase moves along it. Once the pre-mRNA is synthesized, the FACT complex replaces the histones to recreate the nucleosomes.

The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes, which is the RNA polymerase responsible for protein synthesis, takes place 1,000 to 2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is subsequently removed by cleavage during mRNA processing.

Regulation of Transcription

The DNA of prokaryotes is organized into a circular chromosome. Proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are often encoded together in blocks called operons. For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon, and transcribed into a single mRNA. You will learn more about operons and other aspects of prokaryotic and eukaryotic gene expression in BIOL 1C.