Review of gene expression and control
An example of transcriptional regulation is in E. coli. The usual energy and carbon source for E. coli is glucose. When glucose is scarce, lactose can be used instead. Lactose is a disaccharide that must be hydrolysed before it can be utilised. The enzyme that catalyses this reaction is β-galactosidase.
! When the bacterial cell needs to do this, it will express the enzyme. However, it is only produced when lactose is present: this is to avoid energy waste. Regulatory elements control the level of expression of genes that code for proteins.
! Upstream from the genes coding for the enzyme (the lactose operon) are located regulator sequences. The p is the promoter region: this is where transcription enzymes will dock to start the process of transcription. The operator is a regulatory piece of DNA between the promoter and the genes. Upstream from there is a repressor. In normal conditions with no lactose, the cell does not need β-galactosidase. When there is no lactose, the repressor gene will be transcribed and the repressor protein will be expressed. This protein will interact with the operator, binding to it and preventing transcription from proceeding; hence, no β-galactosidase is produced.
There are hundreds of different restriction enzymes and all cut at different sites. They will cut at that site and no other. All of these sites are palindromes (when the opposing strand is read 5’-3’, the same sequence is obtained).
! All of these sites come from different bacteria, as shown. Different bacteria produce a unique restriction enzyme. Note that some only need four bases as their recognition sequence, meaning that they cut much more frequently. Others require six bases. Some cut directly opposite each side on the strand, whilst others make staggered cuts with overhang. The terminology is that they either produce blunt ends or sticky ends. We now have a vast repertoire of tools to cut DNA at very precise sequences. Slight inconveniences arise from time to time because we may not find an RE that will cut exactly where we want them to.
Recombinant technology II The previous lecture contained material on the processes for isolating and manipulating a section of DNA. In order for this operation to work in practice, we must isolate a gene, place it in a vector, sequence it and then express it, move it or use it. The first of these already creates issues. Locating a gene of interest is not at all simple, considering that in a human there are three billion base pairs and only a relatively short sequence must be obtained. First, we need to know what gene we are looking for. Is it a disease gene? Do we know where in the DNA the gene might be located? Is it from a prokaryote or a eukaryote? There are many additional problems if the gene is from a eukaryote. Eukaryotic cells contain a large amount of DNA, which presents an issue, but more importantly, eukaryotic cells manipulate their gene expression in subtle ways, such as splicing out introns and splicing together exons. Pre-mRNA might not be identical to the base sequence required to create the polypeptide of interest. One possible way of dealing with this problem is reverseengineering the sequence of base pairs from the protein. However, this is very difficult to do in the lab. One of the difficulties associated with this is that the genetic code is not one-to-one, meaning that we do not know which codons should be chosen to recruit a particular amino acid. The actual method is the use of mRNA. In the nucleus, the gene with its exons and introns will be transcribed to mRNA and then spliced. Then, the mRNA is released, and it is identified by its 5’ cap and poly-A tail. It can be isolated because of the poly-A tail— nothing else in the cell contains ~200 adenine base pairs in a row. A primer can be made to bind to the A tail and then an enzyme can be used to copy the RNA to turn it into complementary DNA (cDNA). Viruses carry out this process themselves: they carry their genetic information as RNA but may need to convert it to DNA in order for it to be recognised by the cell to be infected. They use an enzyme called reverse transcriptase. An enzyme degrades the RNA, leaving a single strange of DNA, the complementary strand to which can then be synthesised. The result is a double-stranded section of DNA with no introns.
! When the bacterial cell needs to do this, it will express the enzyme. However, it is only produced when lactose is present: this is to avoid energy waste. Regulatory elements control the level of expression of genes that code for proteins.
! Upstream from the genes coding for the enzyme (the lactose operon) are located regulator sequences. The p is the promoter region: this is where transcription enzymes will dock to start the process of transcription. The operator is a regulatory piece of DNA between the promoter and the genes. Upstream from there is a repressor. In normal conditions with no lactose, the cell does not need β-galactosidase. When there is no lactose, the repressor gene will be transcribed and the repressor protein will be expressed. This protein will interact with the operator, binding to it and preventing transcription from proceeding; hence, no β-galactosidase is produced.
There are hundreds of different restriction enzymes and all cut at different sites. They will cut at that site and no other. All of these sites are palindromes (when the opposing strand is read 5’-3’, the same sequence is obtained).
! All of these sites come from different bacteria, as shown. Different bacteria produce a unique restriction enzyme. Note that some only need four bases as their recognition sequence, meaning that they cut much more frequently. Others require six bases. Some cut directly opposite each side on the strand, whilst others make staggered cuts with overhang. The terminology is that they either produce blunt ends or sticky ends. We now have a vast repertoire of tools to cut DNA at very precise sequences. Slight inconveniences arise from time to time because we may not find an RE that will cut exactly where we want them to.
Recombinant technology II The previous lecture contained material on the processes for isolating and manipulating a section of DNA. In order for this operation to work in practice, we must isolate a gene, place it in a vector, sequence it and then express it, move it or use it. The first of these already creates issues. Locating a gene of interest is not at all simple, considering that in a human there are three billion base pairs and only a relatively short sequence must be obtained. First, we need to know what gene we are looking for. Is it a disease gene? Do we know where in the DNA the gene might be located? Is it from a prokaryote or a eukaryote? There are many additional problems if the gene is from a eukaryote. Eukaryotic cells contain a large amount of DNA, which presents an issue, but more importantly, eukaryotic cells manipulate their gene expression in subtle ways, such as splicing out introns and splicing together exons. Pre-mRNA might not be identical to the base sequence required to create the polypeptide of interest. One possible way of dealing with this problem is reverseengineering the sequence of base pairs from the protein. However, this is very difficult to do in the lab. One of the difficulties associated with this is that the genetic code is not one-to-one, meaning that we do not know which codons should be chosen to recruit a particular amino acid. The actual method is the use of mRNA. In the nucleus, the gene with its exons and introns will be transcribed to mRNA and then spliced. Then, the mRNA is released, and it is identified by its 5’ cap and poly-A tail. It can be isolated because of the poly-A tail— nothing else in the cell contains ~200 adenine base pairs in a row. A primer can be made to bind to the A tail and then an enzyme can be used to copy the RNA to turn it into complementary DNA (cDNA). Viruses carry out this process themselves: they carry their genetic information as RNA but may need to convert it to DNA in order for it to be recognised by the cell to be infected. They use an enzyme called reverse transcriptase. An enzyme degrades the RNA, leaving a single strange of DNA, the complementary strand to which can then be synthesised. The result is a double-stranded section of DNA with no introns.
