DNA Transcription

Before we begin our discussion on prokaryotic transcription, it is helpful to first point out some similarities and differences between the process of DNA replication and DNA transcription. The processes that synthesize DNA and RNA are similar in that they use similar nucleotide building blocks. They also use the same chemical method of attack by a terminal -OH group of the growing chain on the triphosphate group of an incoming nucleotide. Both replication and transcription are fueled by the hydrolysis of the pyrophosphate group that is released upon attack. There are, however, a number of important differences between these two distinct processes.

One major difference rests on the fact that while DNA replication copies an entire helix, DNA transcription only transcribes specific regions of one strand of the helix. During DNA transcription, only short stretches (about 60 base pairs) of the template DNA helix are unwound. As the RNA polymerase transcribes more of the DNA strand, this short stretch moves along with the transcription machinery. This process is different from that in DNA replication in which the parent helix remains separated until replication is done.

There are slight differences in the substrates that are used in DNA replication versus transcription. Recall the structural differences between DNA and RNA. RNA's nucleotides are not deoxyribonucleotide triphosphates as in DNA. Instead, they are simply ribonucleotide triphosphates, meaning they do not lack an -OH group. Additionally, in RNA the thymine base is replaced with the base uracil. Both of these differences can be seen in DNA transcription.

Another major difference is that DNA replication is a highly regulated process that only occurs at specific times during a cell's life. DNA transcription is also regulated, but it is triggered by different signals from those used to control DNA replication.

One final difference lies in the capabilities of RNA polymerase versus DNA polymerase. Remember that a key problem in DNA replication lay in the initiation of the addition of nucleotides. RNA primers are needed to begin replication because DNA polymerase is unable to do it alone. DNA transcription does not have the same problem because RNA polymerase is capable of initiating RNA synthesis. The structure of the RNA polymerase is necessary for understanding all of the processes that underlie initiation, elongation, and termination and also explain some of its added capabilities.

There are two main segments of the RNA polymerase molecule: the core enzyme, and the sigma subunit. These two pieces are together referred to as the "holoenzyme". The core enzyme is itself composed of a beta, beta prime, and two alpha subunits; together the core is responsible for carrying out the polymerization or synthesis of RNA. The sigma subunit of RNA polymerase is the part of the enzyme responsible for recognizing the signal on the DNA strand that tells the polymerase to begin synthesizing RNA. It is through this sigma unit that RNA polymerase is able to initiate transcription.

In prokaryotic cells, free RNA polymerase molecules are constantly colliding with DNA helices. The collision leads to a weak association between the DNA and RNA polymerase, which is soon broken. However, when the RNA polymerase binds to a specific sequence on the DNA, it binds tightly, forming a DNA/RNA polymerase complex. This specific site of binding is called the start site. The start site represents the location on the DNA that marks where the first nucleotide of an RNA chain should go; that spot is designated as the "plus one position". Positions that are designated as downstream in the RNA are positively numbered according to their relative position to the plus one position. All positions designated as upstream of the start site are labeled with negative numbers according to their position relative to the start site. Sequences located just upstream of the start site, called the promoter region, contain the information that signals the RNA polymerase to start transcription.

There are a number of key features to the promoter region that give it the ability to provide the signal initiating transcription. While nearly all promoters vary slightly, they all have general traits that can be identified. Located approximately 10 and 32 base pairs upstream of the start site are two such regions, called the -10 and -35 sequences. Each sequence consists of six base pairs. For an ideal promoter, the sequence is TTGACA for the -35 region and TATAAT for the -10 region.

In addition to the specificity of the bases in these sequences, the spacing between the two is also important. Ideally, this gap is 17 base pairs long. Deviations from this spacing have significant effects on the strength of the promoter region. The closer a promoter region is to matching this canonical promoter sequence, the greater its strength.

There is a third promoter element that is sometimes seen in very strong promoters which is called the UP element. It usually is composed of alternating stretches of 5 adenine and thymine bases. It is located upstream of the -35 region.

RNA polymerase binds to the DNA helix at the start site. Bound to DNA, it covers a 60 base pair region within which it scans for the -35 and -10 promoters. Initially, the polymerase, and specifically the sigma subunit, binds in what is called a "closed complex" to the DNA. The RNA polymerase/promoter complex then undergoes a conformational change that breaks a number of base pairs extending from the -10 region to create a bubble in which the two DNA strands have separated. This bubble is usually approximately 17 base pairs in length. This new formation is called the "open complex". RNA synthesis is then initiated using one of the DNA strands as a template for adding complementary RNA base pairs. Transcription is usually initiated with a purine, rather than pyrimidine, base. Once initiated, the RNA polymerase moves down the DNA strand in the elongation process, which is covered in the next section.