The total energy budget of a prokaryote cell with its 4.6 million nucleotides and 4,288 different genes, is about 10 billion ATP. This is significant. The cell must therefore make sure that this massive expenditure is wisely used by making the right genes at the right time in the right amounts. This article will explain how this is achieved.
RNA Polymerase Subunits
First, let’s explore the structure of the RNA polymerase in prokaryotes. The structure is a modular one consisting of 6 subunits that includes 2 alpha, one beta, one beta prime, one omega subunit, and a sigma factor. All the subunits together, except the sigma factor, is referred to as the core enzyme. When the sigma factor is attached, it is referred to as the holoenzyme.
- The two alpha (α) subunits hold the subunits on the holoenzyme together
- The beta (β) subunit brings in nucleotides to make a new mRNA copy
- The beta prime (β’) attaches to the DNA template strand
- The function of the ω subunit is unclear
The sigma factor is the part of the RNA that binds to the DNA at the -35 and -10 promotor sites. Thus, the attachment of the sigma factor allows initiation. Once attached, the holoenzyme denatures 14 nucleotides around the promotor and transcription begins. This state is known as the open promotor complex.
Transcription cannot be followed through to its completion until the sigma factor falls off the complex. In cases where the loss of the sigma factor is delayed, the next steps can be a series of abortive initiations in which the RNA polymerase attempts and fails to transcribe the gene. The time it takes for the sigma factor to clear the RNA polymerase is referred to as the promotor clearance time. This in some cases can take several seconds, and hence can be a rate limiting step in transcription. Once the sigma factor is removed however, the RNA polymerase is no longer stuck to the promotor but can move along the DNA to transcribe the gene.
Location of RNA Polymerases
Controlling all the transcription that needs to be accomplished for all of the over 4,000 genes in an E. coli, are about 7,000 RNA polymerases. These enzymes are not all just floating around in the cytoplasm but are instead physically associated with the DNA molecule. The attachment is facilitated by the electrostatic interaction between the negatively charged DNA and positively charged protein subunits of the RNA polymerase. Physical attachment, allows the RNA polymerase to find a promotor quickly by moving up and down the length of the DNA in a 2-dimensional search rather than a 3-dimensional search if it was detached. About 1,500 RNA polymerase enzymes are involved in this type of loose binding.
When RNA polymerase picks up a sigma factor, it binds tightly to the DNA. At any point in time, there are about 1,000 RNA polymerases engaged in tight binding. Remember that the attachment of the sigma factors forms the holoenzyme and that the function of the holoenzyme is to find a promotor. Once it does that, the DNA is denatured and opens to start transcription. About 1000 RNA exists in this open promotor complex.
The remaining 3,500 of the 7,000 RNA in E. coli, exists in the ternary complex form. This is the state where the DNA is being transcribed to form mRNA. Ternary refers to the three parts of the complex, i.e., core enzyme, DNA, and the nascent (new) RNA.
Switching Out Sigma Factors
Remember that it is the sigma factor that sits on the promotor site of the DNA to initiate transcription. There are 7 forms of the sigma factor. The modular design of the RNA polymerase permits them to be switched out for one form or the other.
The housekeeping sigma factor (the one that is used under normal circumstances) is referred to as σ70 since it has a molecular weight of 70 kDa. Its job is to look for the TTGACA sequence at the -35 promotor site and the TATAAT sequence at the -10 promotor site. This is perfect for general use, however, under circumstances such as stress, the cell may need to produce new proteins to cope and survive. In this case, the σ70 module may be switched out to accommodate a more functional sigma factor.
Below is a list of the 7 sigma factors, their use, and promotor sequences.
Prokaryotic Promotor Strength
Prokaryotes like E. coli can achieve additional functionality by modulating the strength of their σ70. This is achieved by the ability of σ70 to recognize sequences that deviate slightly from its consensus sequence, i.e., TTGACA and TATAAT. Although it recognizes these other sequences, it does so at weaker strengths. This ability enables E. coli to transcribe the same genes at different rates as needed.
Prokaryotes can avoid squandering of energy expenditure by determining what type of genes needs to be transcribed given the situation. For example, under stress, it can turn off its housekeeping sigma factor and turn on alternate sigma factors to make proteins that will respond to the stress. They also have the ability to control the rate of transcription of housekeeping genes by σ70 being able to recognize different sequences that deviate slightly from its consensus sequence.
Reference: Krane, D. 2021. Bio 2110 Molecular Biology Video Lecture. Wright State University – Lake Campus.