The first step toward gene expression is the transcription of a DNA template into a complementary RNA strand. This process is done by RNA polymerase, which reads the DNA template and produces an antiparallel RNA copy. As in DNA replication, the complementary strand is produced 5'->3'If the DNA template encodes for a gene, this RNA transcript will be refined into mRNA, which is further translated into a functional protein. The RNA transcript may also go on to make ribosomal RNA (rRNA), transfer RNA (tRNA), or many other RNA products. The entire process can be broken into three major steps: initiation, elongation, and termination.
Initiation of transcription occurs differently in eukaryotes and prokaryotes. In eukaryotes, the transcription initiation complex must be formed. This includes, the core promoter, transcription factors, RNA polymerase, and activators/repressors. In E. coli, RNA polymerase and sigma factors are needed, as well, it may be necessary to have activators/repressors based on the promoter being used. For E. coli, the RNA polymerase will bind tightly to the promoter to form an open promoter complex, then must choose the transcription start site and escape from the promoter. It is necessary to balance the strength of promoter binding the ability to escape so elongation can happen. RNAP may undergo abortive initiation in which it will form many short 9-10 bp segments until it clears the promoter and begins elongation.
A DNA sequence that recruits transcriptional machinery and lead to transcription of downstream DNA. In E. coli the -10bp and -35bp are locations of the most well conserved DNA sequences in bacterial promoters. There are on average 17 bp between the two sequences and 7 bp between the -10bp location and the transcription start site. Consensus sequences are the nucleotide sequences that share a common function, which is binding to RNAP in the case of promoters. Promoters that most closely resemble the consensus sequence will be the strongest promoters, just as those that differ from the consensus sequence will be weaker promoters. Interestingly, there has not been a promoter found in E. coli that is of the consensus sequence, it would likely bind so strongly that elongation would not occur.
An unregulated promoter that allows for continual transcription of its associated genes. These promoters do not rely on input and depend only the level of free RNA polymerase holoenzyme are referred to as constitutive. Since the holoenzyme is needed, it can also be said that these rely on the level of sigma factors.
Positive, Negative, and Multi-regulated promoters
These promoters depend on the level of transcription factors that are not sigma factors. In positively regulated, as the concentration of activator increase, the rate of transcription also increases. If an activator protein relies on the binding of an exogenous molecule to activate it, then the promoter may be referred to as inducible. For negative promoters, increased levels of a repressor will lower the activity of these promoters. If a repressor that inactivates the promoter is always present and an exogenous molecule is added that binds the repressor and deactivates it, then promoter may be referred to as inducible. Multi-regulated promoters are either positively or negatively regulated by multiple transcription factors. These are most useful when a promoter that relies on multiple environmental factors to function is desired.
Prokaryotic Sigma Factors
The E.coli RNA polymerase consist s of 5 subunits:2α, β, β', ω. The sigma factor is the 6th subunit, it is needed in forming the RNAP holoenzyme complex which is necessary in promoter binding. The sigma factor helps to recognize the -10 and -35 bp segments of the promoter. The most common sigma factor used in E. coli is the σ70 subunit. This is the housekeeping sigma factor and is used during transcription of most genes. It recognizes the consensus sequence: TTGACA__(17)__TATAAT. There are an additional 6 sigma factors, active in different situations. Such as σ32, which is the heat shock sigma factor. The B. subtilis housekeeping sigma factor is σA, similar to σ70 in E. coli.
The strength of the different promoters is determined by the relative frequency of transcription initiation. This is mainly affected by the afﬁnity of the promoter sequence for RNA polymerase. (cite) Promoters that differ significantly from the consensus sequence will be weaker than those that resemble the consensus sequence due to binding affinity.
E. coli Promoter: LacUV5
LacUV5 is a mutated form of the lac promoter used in E. coli. The lac promoter is considered weak, it varies from the consensus sequence by 3 bases. On the other hand, the lacUV5 mutated promoter varies from the consensus sequence by only 1 base and is much stronger than the lac promoter.
Strong Promoter: Bacteriophage T7 Promoter
The T7 promoter is derived from bacteriophage T7. The T7 RNA polymerase has a very high affinity for its own promoters which do not occur naturally in E. coli. In the experiment done by moffatt et al. the gene transcribing T7 RNAP was introduced under the control of the lacUV5 promoter. They showed that the T7 RNAP will transcribe almost any gene connected to a T7 promoter introduced into the E. coli genome. It was found that the mRNA transcripts were saturating the translational machinery of E. coli. A target protein could accumulate up to 50% of the total cellular protein in ~3 hours.
#$% Yeast Promoter
In this type of termination, a protein factor called Rho destabilizes the DNA template-RNA transcript complex, causing the release of the RNA transcript. Rho-dependent terminators are not included in the iGEM registry because these terminators are not specified by sequence.
The terminators are composed A, T rich sequences as well as a two-fold symmetric DNA sequence. When transcribe by RNA, these sequences lead to a hairpin loop rich in G-C base pairs followed by many bases of uracil. The formation of the RNA G-C rich stem loop causes a pause in the RNA Polymerase. This pause, followed by the transcription of the poly A tail into a run of U's causes a mechanical stress and the unwinding of the RNA-DNA complex, causing the dissociation of the RNA transcript from RNA polymerase.
#$% Bacterial Terminator
In yeast, termination is different for each RNA polymerase (I-III). The process involves the polyadenylation at the 3' end of the RNA transcript. A set of proteins cleave off the RNA transcript and then synthesize the poly A tail, independent of the DNA template. This step is important toward refining the RNA into mRNA that will translated.
#$% Yeast Terminator
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