8.3: Protein synthesis- transcription (DNA to RNA)

Having introduced DNA, mRNA, and the genetic code, we turn to the process by which a polypeptide, specified by a DNA sequence, is synthesized in a cell. First how is the specific DNA region that encodes a specific polypeptide recognized. We are looking for a relatively short region within millions (in prokaryotes) or billions (in eukaryotes) of base pairs of DNA. This information will be present in one strand of a double-stranded region of DNA. From the point of view of polypeptide synthesis, the other strand is effectively nonsense. This means that a gene’s regulatory sequence must specify which strand is used to generate a mRNA, as well as where, when and how often RNA synthesis starts.

To "find" a gene a specific nucleotide sequences is used, together with something (a protein) that recognizes and binds to that specific nucleotide sequence. Let us consider a particular form of the problem; we want to uniquely specify one gene (one sequence) within the ~3,000,000 base pairs of an E. coli cell's genomic DNA. For simplicity let us assume that the A:T ratio equals the G:C ratio. Clearly a one base pair sequence will not work, since we might expect that half of the base pairs will be recognized, either directly as T or indirectly as A bonded to T on the other strand. To be unique the sequence we want must occur once in 3,000,000 base pairs (1/3,000,000 = 3.33… x 10^-7 = 0.000000333). If we use a two base sequence, it will occur 1/4 x 1/4 = 1/16 = 0.0625, a four base sequence 0.0039, an eight base sequence 0.00001523, but a 16 base sequence has a probability of occurring purely by chance of ~2.32 x 10^-10, which is less than once per genome.^[330]

There are DNA sequence specific binding proteins that acts to control transcription, they are known generically as transcription factors. Positively acting transcription factors act to recruit DNA-dependent, RNA polymerase to a specific site and strand on the DNA, upstream of a gene's coding region. A negatively acting transcription factor can block the activity of positively acting factors. An positively acting transcription is the first step in gene expression (→). The combination of transcription factors present and their activity in a cells will determine gene activity.

Gene specific RNA synthesis is mediated by DNA-dependent, RNA polymerase, encoded by specific genes. As in the case of DNA-dependent DNA synthesis, RNA synthesis is also subject to errors; estimates for the error rate range from 10^-3 to 10^-6 errors per nucleotide incorporated.^[331] Where, and in which orientation, the polymerase binds to the gene’s DNA is determined by the gene’s regulatory sequence(s) and the orientation of the proteins bound to it. Which genes are expressed is determined by which transcription factors are present and active, their concentrations, and their binding affinities for specific regulatory sequences, as well as the presence of competing binding sites. Polymerase binds and is activated through binding to a gene's DNA-transcription factor complex. Since there are many genes in the genome, the stability of the DNA-Transcription Factor-Polymerase complex will impact the number of RNAs synthesized per unit time. A similar process is used for genes that encode "non-coding" RNAs.

At this point, it is useful to explicitly recognize some common aspects of biological systems. These systems are highly regulated, adaptive, and homeostatic; they adjust their behaviors to changes in their environment (both internal and external) in order to maintain the living state. These behaviors are based on various forms of feedback regulation. In bacterial gene expression, there are genes that encode specific transcription factors. Which of these genes are expressed, together with the half-lives of transcribed RNAs and encoded proteins determines the concentrations of the various transcription factors present and, in turn, the pattern of gene expression. Of course, the gene(s) encoding a specific transcription factor are regulated. Transcription factors can act positively or negatively; they can lead to the activation of transcription by recruiting and activating the RNA polymerase or by blocking its recruitment and/or activation. In addition the activity of a particular transcription factor can be regulated. Please note that most transcription factors are involved in the regulation of multiple (often hundreds or more) genes, can act either positively or negatively, and that their binding affinity for the regulatory sequences of different genes can differ.

All organisms are complex. A “simple” bacterium contains thousands of genes and different sets of genes are used in different environments and situations, and in different combinations to produce specific behaviors. In some cases, these behaviors may be mutually antagonistic. For example, a bacterium facing a rapidly drying out environment might turn off genes encoding proteins involved in growth and turn on genes to prepare to survive in a more hostile environment. Our goal here is not to generate perfectly accurate predictions about the behavior of an organism in a particular situation, something that may not be possible without specific (provided, memorized) information, but rather to help you consider plausible models for how gene expression might change in response to various perturbations. This requires us to consider, although at a elementary level, the regulatory processes active in cells.

For a transcription factor to regulate a gene, whether positively or negatively, it must be able to bind to specific DNA regulatory sequences with a reasonable affinity, meaning that it stays bound long enough to recruit (or inhibit) the binding and activation of other factors, concluding with RNA polymerase, before being knocked off by thermal collisions. Groups of genes that are expressed together, in response to common cellular, developmental, or environmental conditions, are likely to be regulated by common sets of transcription factors. The activation or inactivation of a transcription factor can involve a number of mechanisms, including its modification or interactions with other proteins that alter its ability to interact with its target DNA sequence, other proteins, or RNA polymerases.

Transcription factors are synthesized in the cytoplasm. In prokaryotic cells, without a nucleus, it can bind to its DNA targets. In eukaryotic cells it first has to enter the nucleus before it can bind to its target DNA sequences. When an RNA polymerase collides with the DNA-transcription factor complex, it can remain bound and initiate the process by which the RNA polymerase is activated and begins RNA synthesis. Once RNA polymerase has been activated, it will move away from the transcription factor-DNA complex. The DNA bound transcription factor complex can then bind another polymerase or it can disassemble, releasing the transcription factors from the DNA (in response to molecular level collisions). Once released transcription factors can diffuse away, interact with other regulatory factors, or rebind to other sites in the DNA. Clearly the number of copies of a particular transcription factor protein present (its concentration), its target site affinity, and the frequency of its various DNA binding sites will impact the behavior of the system, as will the concentrations of ancillary factors that interact with the transcription factor/DNA complex to recruit and activate the polymerase.

RNA synthesis is a thermodynamically unfavorable reaction, so for it to occur it must be coupled to a thermodynamically favorable reaction, in particular nucleotide triphosphate hydrolysis reactions. The RNA polymerase moves along the DNA to generate an RNA molecule (the transcript). Other signals within the DNA, and recognized by proteins associated with the transcription machinery, lead to the termination of transcription and the release of the RNA polymerase. Once released, the RNA polymerase returns to its inactive state. It can act on the same or another gene if the RNA polymerase interacts with transcription factors bound to the gene’s promoter. Since multiple types of transcription factor proteins are present within the cell and RNA polymerase can interact with all of them, which genes are expressed within a cell will depend upon the relative concentrations and activities of specific transcription factors and their regulatory and associated proteins, together with their binding affinities for specific DNA sequences compared to their general low-affinity binding to DNA.

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