How do cells store, intraconvert, and interpretpret information?  Organisms grow and develop while their bodies are regulated and maintained so that they can successfully reproduce in order to form a new generation.  How do all these processes function?  Nucleic acids and proteins play the roles of information molecules in cells.  Here are a few more questions worth pondering before we delve into the information flow of cells.

What is the basic nature of information representated by these molecules? (We will see the importance of linear sequences and three-dimensional structures)
How is information converted from one form to another?
How is the information selectively converted? (In other words, how is the linear nucleotide gene sequence expressed into animo acids?)
What kind of vocabulary can we develop to enable us to describe and discuss information molecules?
How can we exploit experimentally what goes on during information flow?

The occurrence of membrane bound organelles for eukaryotes (more specifically, the nucleus) creates a spatial and temporal difference.  In other words, transcription occurs in the nucleus while translation occurs in cytoplasm, and transcription/translation may proceed at different times since they are UNCOUPLED.  This uncoupling allows for eukaryotes to increase the quality of the process by enforcing more controls (i.e. cutting out intons) to reduce the amount of errors.

RNA polymerase II and general transcription factors (GTFs) contribute to transcription.  The primary transcript is the initial UNPROCESSED single strand molecule.  Transcription begins at the 5' end where a methyl guanosine cap is created.  This cap site will 1)stop degradation of the 5' RNA strand, 2)make nuclear export more efficient, and 3)make initiation of translation of mRNA more efficient as well.

Now on the 3' end, processing has 2 steps that will "snip off" this edge of the strand at specific points by endonucleases.  1)Cleavage removal of a stretch of nucleotides at the 3' end is performed by endonucleases and other helpers.
2)20 - 250 adenines are added by polyA polymerases to create the "poly A tail."

The poly A tail will increase RNA stability and translatability.

Now the mRNA, consisting of only exons, must leave the nucleus through NUCLEAR PORES. 

Remember the uncoupling of transcription and translation mentioned earlier?  What else can these extra steps do to increase efficiency of gene expression?  Well, the many steps involved allow the cell to control  various mechanisms and products at different points along the way.  Proofreading, switches, delays, and other maneuvers enhance the specificity of the whole process.  The numerous steps are responsible for a large amount of proteins so that MORE CAN BE DONE to improve gene expression.

Gene expression can be controlled at various stages.

1)Control at the level of transcription
        There are 2 important regions of a gene, upstream promoter sequences and coding sequences (where information for protein structure and function lie).  In the promotor sequences, RNA polymerase II binds to the TATA box (initiator of transcription) by the aid of TATA-binding proteins (TBP).  DNA-bound GTFs interact with subunits bound to TBP so that DNA will loop around.  Protein-protein interactions increase transcription performance so enhancers, which can be tens of thousands of base pairs away (and work upstream, downstream, inverted), will promote the assembly of the basal transcription machinery at the promoter and interact with activators and coactivators.  The CAAT and GC boxes bind transcription factors which aid in polymerase efficiency.  Transcription factors can be stimulated (by glucocorticoids) or inhibited (by negative regulatory proteins that bind to promoter elements) and affect the regulation of the basal level of transcription (which is initiated by promoters).  Methylation can inactivate gene expression, and removal of acetyl groups can repress transcription.  Another way to alter proteins is to cut pieces of promoters.  For instance, blue cotton could be created.

2)Control at the level of RNA processing
        Alternative splicing allows for a gene to encode for two or more related proteins.  This is the reason parts of an intron in one cell can be part of an exon in another cell.  An example of control at the level of RNA processing is the shifting of the poly A site to control the length of the mRNA strand.

3)Control at the level of RNA editing
        The nucleotide base, cytosine (C), can be converted into uracil (U).  ACG codons can create start AUG codons.  A strand can be shortened when editing CAG, CAA, and CGA codons to produce stop codons.

4)Control at the level of RNA transport
        If mRNA can't get out of the nucleus, it can't be translated into a protein.  As we talked about earlier, the methyl guanosine cap and poly A tail help in RNA nuclear export and stability, respectively.  Adenoviruses will use this control to prevent host transport, but still allow for viral genes to continue onto translation.  HIV and influenza inhibit host mRNA as well.

5)Control at the level of translation
        This occurs in the cytosol.  The untranslated region on the 3' side contains information for the localization of mRNA.  For example, the bicoid gene is localized at the anterior end of the oocyte because it helps in head and thorax development.  The oskar gene is at the posterior end for germ cell formation.  Also, an unfertilized eggs will have inhibitory proteins to mask mRNAs for developmental use.  Ferritin is a protein that grabs iron atoms.  At low iron concentrations, the repressor of ferritin's mRNA is active at the 5' end.  When there is more iron around, repressor is modified and it loses affinity for the sequence (found in the untranslated region) for it usually binds.  The control on ferritin is lifted now so it can detoxify the cells.

6)Control of RNA stability
        Half life is the time for half of the population to degrade.  If proteins live longer, there will be a larger population even though production of each strand is accomplished at the same rate.  More mRNAs will be available to act as templates.  In prokaryotes, the 5' end begins degredation before the 3' end is even comopleted.  mRNAs that are used for short periods (i.e., cell division) will be have short lives and those used for the production of dominant proteins (i.e., hemoglobin and ovalbumin) live longer.  Nucleotide sequences in the 3' untranslated region have CCUCC repeats that allow for proteins to bind and stabilize the poly A tail while poly A nucleases eat away at it.  AU-rich sequences will destabilize the tail.  Note that the loss of the 3' tail will allow for degradation to begin at the 5' end which will eventually move to the 3' end.

Differences in gene expression can be studied by looking at the proteins created.

    Proteins can act as an antigen for an antibody.  The use of indicator dyes and possibly primary and secondary assays would allow for detection of specific proteins.

An easier way would be to focus on the mRNA that make the proteins because of various "tricks" that can be implemented.

    Two dimensional gels can use molecular weight and isoelectricity to identify the exact gene in question.  A metabolic label can be placed on the specimen by radioactivity.  The gene can be cut out of the gel so that one may work backwards to work on finding the amino acid sequence.
    Single stranded probes are used to in hybridization.  The known identity of the probe allows for isolation of preferred mRNA strands.
    To find a specific gene (or all of them), a cDNA library will come in handy.  Reverse transcriptase will convert mRNA into complimentary DNA (cDNA).  The double stranded version of this can be incorporated into a cloning vected (i.e. plasmid) and inserted into bacteria.  Dilution plating will allow for each separate colony to consist of different mRNA.  This library can be probed.  The cDNA library will only contain the coding sequences and not the upstream regulatory sequences.  Another problem is that since promoters direct the timing and extent of gene expression, their absence allows for bias.  If the cDNA in the library is not representational of the actual amount, expression could range from "cowabunga" to just a little bit.
    To obtain the regulatory sequences, one must start with genomic DNA.  Enzyme restriction digests will cut DNA into pieces.  Single stranded versions can be inserted into plasmids.
    One genome project, total expression monitoring, involves the representation of known nucleotide sequences on an array created by dual hybridization of cDNA.  Recently Dr. William F. Loomis from the Center for Molecular Genetics, University of California-San Diego, spoke at Texas Tech University .  He talked about silicon chips that contained entire cDNA libraries.  Techniques in the field of genetics and cell biology continue to facilitate research as scientists delve into the new millennium.