Gene Expression II

In a lytic cycle, the phage replicates until the cell lyses and the progeny phage are released. If the cell is growing rapidly, it benefits the phage to have a lytic cycle so that it can infect more cells immediately after producing progeny. In a lysogenic cycle, the phage DNA integrates itself into the host cell DNA where it is called a prophage. The decision of which cycle to utilize is based on physiological conditions of the cell and its environment.

Eukaryotic genes are "off" until something active triggers them to be expressed. Totipotency refers to the fact that all nuclei in an organism contain the same set of genetic information, even though not all the information is used in any one cell. This was demonstrated in an experiment, in which an unfertilized frog egg was treated with UV light which inactivated the nucleus of the egg cell. The nucleus of a skin cell from a tadpole was then isolated and injected into this egg cell. In a number of cases the nucleus from the tadpole skin cell allowed the egg to develop normally. This experiment proved that all the genetic information is present in any cell but is selectively expressed in each. No genetic information is lost during development, but the set of genes expressed in different cell types changes.

For gene expression, prokaryotes require RNA polymerase, promoters, termination sites, and operators. Eukaryotes require three RNA polymerases.

• RNA polymerase I synthesizes ribosomal RNA (rRNA is 90% of RNA in cell) and needs accessory factors to initiate transcription (positive control factors). RNA polymerase I appears to be species specific because rRNA from one species often can not be transcribed by the RNA polymerase I from another species.
• RNA polymerase II synthesizes pre-messenger RNA (pre-mRNA). It is the most delicately regulated eukaryotic RNA polymerase, it needs accessory proteins (transcription factors) and is not species specific.
• RNA polymerase III is responsible for the synthesis of tRNA, 5S RNA and snRNA (small nuclear RNA).

• In a testube containing DNAs with 5S genes and flanking sequences, add RNA polymerase III, ATP, CTP, GTP, UTP, and any other necessary factors for transcription. This results in synthesis of RNA that corresponds to 5S RNA. This type of experiment is called in vitro transcription.
• Trim off some of the 5' flanking region of the DNA ("upstream" of the gene itself) and repeat the in vitro transcription.
• As flanking region is trimmed, eventually the promoter will be cut out and transcription will not occur because the RNA polymerase cannot initiate transcription. Researchers found that it was possible to delete the entire 5' flanking sequence and still get 5S RNA synthesized.
• The researchers continued and actually deleted the 5' end of the 5S DNA itself (and substituted it with other DNA). Remarkably, initiation still occurred in the right location.
• Eventually it was determined that there was a region in the middle of the 5s gene (nucleotides 55-80) that is actually the promoter.

It is thought that the RNA polymerase binds to the promoter (or binds to transcription factors at the promoter) and the DNA wraps around the ball of RNA polymerase and begins transcription at the appropriate location on the DNA.

To regulate the level of 5S RNA in the cell, in this case, a specific transcription factor can bind to either the 5S RNA or to the 5S gene responsible for its synthesis. If transcription factor binds to the RNA, as more product is made, there will be less transcription factor available to bind to the promoter so transcription of the 5S gene will slow down. If there is little 5S RNA around, then most of the transcription factor will bind to the 5S gene and stimulate transcription of the 5S RNA. This is a feedback system.

For RNA polymerase I and usually for III, there exists a specific termination signal (sequence of nucleotides) on the DNA. For RNA polymerase II, however, there is no specific termination sequence. The RNA polymerase continues until it "falls off," then the precursor RNA is trimmed at a specific site to produce the final pre-mRNA product.

When the RNA is synthesized a cap is added to the 5' end of the RNA. This specific unique structure interacts with eukaryotic ribosomes to help initiate translation. Since the cap is needed for initiation of translation, a polycistronic eukaryotic mRNA will not function because the cap can only reside at the 5' end of the RNA - the middle cistrons cannot be translated because they do not have a nearby cap. A string of adenosines called the poly(A) tail is added to the 3' end of pre-mRNA. Eukaryotic pre-mRNAs also contain introns (for intervening sequences), which are sequences between coding sequences (called exons for expressed sequences) that don't code for anything. The process of RNA splicing removes introns to create mature mRNAs ready for export from nucleus to cytoplasm.

Methyl groups attach themselves to CG (cytosine-guanine) doublets on DNA. Extensive methylation near a gene will generally prevent transcription of that gene. If the methyl groups are removed, transcription can, but not necessarily will, occur again. In other words, the absence of methylation is a necessary (but not sufficient) condition for transcription to occur.

No demethylase enzymes have been found; that is, no enzyme has been discovered that can remove the methyl groups once they become attached to the DNA. The only way methyl groups can be removed is through the creation of new strands, which are not methylated at first - this only occurs during DNA replication. There are a few consequences to methylation: preventing methylation could turn genes on and off, could create tumors and other problems. Methylation cannot be a universal method of regulation gene expression because at least some organisms (like fruit flies) don't methylate their DNA.

One gene product might be useful to a cell in a number of different situations. A gene which is multiply regulated like this would have to be able to be turned on by a number of different methods corresponding to the set of stimulating circumstances. For example, a gene that might help a cell survive stressful conditions needs to recognize changes in the environment which might be the result of low oxygen levels, the presence of toxic substances (e.g. heavy metals or alcohol), or even a fever (elevated temperature) within the organism itself. Such a gene having multiple functions can't be regulated with only one operator, but requires a variety of control elements with which it can respond to different stimuli. A number of transcription factor binding sites (might be different elements of a promoter) are upstream of the gene and the level of transcription depends on which transcription factors are present to bind to those signals.

Each element of the promoter might be capable of recognizing a few different transcription factors and transcription factor is capable of recognizing a number of promoter elements. If more than one transcription factor can bind to a particular promoter element, the different transcription factors will compete for binding to that element. The level of transcription from the regulated gene will depend on the relative levels of the different transcription factors. If one transcription factor strongly recognizes promoter element B but also weakly recognizes promoter element A, you have a system that is capable of delicately regulates transcription level.

Some sequences don't seem to be connected with the promoter of a gene but still can influence the level of transcription from that gene. These elements, called enhancers, exists elsewhere on the DNA but must reside on the same DNA molecule to work. Enchancers, which can point in either direction, can be tens of thousands of basepairs away from the gene and can be present upstream, downstream, or even internally with respect to the gene it is regulating. Enhancers can increase the level of transcription by stimulating transcription from all nearby genes. Some transcription factors can also recognize and bind to enhancer elements. This allows for additional control on broader level, providing control of a set of connected genes.

Problems can arise if enhancers are where they shouldn't be, for example: viruses can have enhancers in their genome that when inserted near a gene, turn on a promoter and express a gene that wasn't being expressed previously.

Isolation of Globin Gene

Most of the mRNA in red blood cells is responsible for the synthesis of hemoglobin. Because of the preponderance of globin mRNA in red blood cells, scientists were able to isolate and purify globin mRNA for study. When globin DNA was isolated and hybridized to globin mRNA a unique structure resulted. There was a large loop of DNA that wasn't base-paired to the purified mRNA. The large loop was an intron that had been spliced out during transcription because it didn't code for anything. Most eukaryotic genes have multiple introns, which has an evolutionary advantage.