Gene Expression and Regulation

As we have just discussed, the structure of DNA provides a mechanism for self-replication. The structure of DNA also reveals the mechanism for storing the genetic information that determines what a cell is and how it functions. This information is known as the genetic code. In this section, we will look at how the information stored in molecules of DNA is used to direct the synthesis of protein. It is also important to know how DNA is regulated in organisms, so that the appropriate DNA is expressed in each cell. We will also briefly look at some of the ways in which gene expression is regulated.

Learning What a Gene Does
Before we discuss how DNA does its job of storing and using genetic information, let's look a bit at one bit of research that led to the conclusion that a gene is a piece of DNA that specifies the amino acid sequence in a polypeptide (or protein).

Beadle and Tatum and Pink Bread Mold
In the 1940’s Beadle and Tatum induced mutations (changes in the genetic code) in Neurospora, a pink mold common on bread, and tracked the metabolism of the mutant strains. They mapped chromosome locations of the mutant strains, and then related their chromosome maps to the presence of absence of specific enzymes needed in Neurospora's metabolic pathway for the synthesis or arginine. From their research, Beadle and Tatum postulated the one gene-one enzyme theory.

Eventually, we also learned that not all genes must code for enzymes; some code for structural proteins or functional proteins (such as the membrane proteins). Furthermore, quaternary proteins are composed of more than one polypeptide, so the concept of gene has been further refined to be that a gene codes for a polypeptide.

But what is the relationship between DNA and genes and protein synthesis in cells?

The expression of DNA in the genetic control of the cell - Preview


Translation requires the interactions of 3 different types of RNA, which convert the original DNA code word instructions (the triplet sequences) into specific polypeptides (unique sequences of amino acids)

However, before we go further in our discussion of transcription and translation we need to look a little more closely at the structure and types of RNA and at the genetic code.

Structure of RNA Molecules
RNA is composed of
Molecules of RNA are single-stranded. However, some RNA molecules fold back on themselves at places, called hairpins, forming complementary base pair bonds.

The Types of RNA
1. Messenger RNA (mRNA)


2. Ribosomal RNA (rRNA)

3. Transfer RNA (tRNA)

The Genetic Code: DNA and RNA at Work - Overview
The information of DNA is coded into three-nucleotide long sequences (a triplet code). Each triplet sequence of nucleotides in a DNA molecule is a "code word" for one specific amino acid. DNA molecules contain a linear sequence of triplets that will specify which amino acids a protein will contain, and the sequence, or order, in which these amino acids will peptide bond to form a polypeptide. Moreover, the code is non-overlapping and there are no separators, or punctuation, between the triplets.

The DNA sequence for a single protein will have three times the number of nucleotides as the number of amino acids in the protein for which the sequence codes. In addition, there will be start and stop regions of the DNA associated with these instructions for protein synthesis, and regions within the DNA molecule that do not code and are removed by RNA processing after transcription.

Although there can be 64 different DNA code words, three of them are "nonsense" and do not code for specific amino acids. The three "nonsense" code words specify the end of a polypeptide coding.

A mRNA nucleotide triplet (synthesized from a DNA template) that codes for a specific amino acid is a complementary (rather than identical) codon to the DNA. Synthesis of RNA follows the same nitrogen base pair rules that dictate DNA replication. Each mRNA transcript will be a faithful, but complement copy of the nucleotides of the DNA template.

The codons for each of the amino acids are known, as well as specific codons which are used as start and stop messages. (Your text has a discussion about how this was accomplished.)

When you look at Codon tables, you will see that some amino acids are coded for by more than one codon. Often, only the first two nucleotides of the triplet are essential; the third is redundant. (e.g., CCU, CCC, CCA and CCG all code for the amino acid, proline, and UCU, UCC, UCA and UCG all code for the amino acid, serine.) But the codon always contains the entire triplet. The reverse is not true. One codon can never code for more than one specific amino acid. UCU codes for serine. UCU can never code for any other amino acid.

The process of translation requires triplet sequences of tRNA that match the mRNA for the amino acid assembly. The only way to match nucleotides is by base pairs, which are complements to each other, so the tRNA triplet that codes for and attaches to a specific amino acid is often called the anticodon.

Each tRNA has an amino acid binding site that can attach to its specific amino acid. Specific enzymes do this. These attachment sites are also phosphorylated (using ATP) to provide the energy for protein synthesis.

Codon-anticodon (mRNA-tRNA) matches occur at ribosomes where the amino acids, which are attached to the tRNA molecules, can be joined by peptide bonds to form polypeptides. Several ribosomes can function at one time so that several copies of a polypeptide can be made at one time.

How it works: Details of Process of Transcription
RNA synthesis uses DNA as a template, and occurs in the nucleus. There are three stages in transcription: Initiation, Elongation and Termination.

Initiation


Elongation


Termination

Note: The process of transcription is also used to synthesize the tRNA molecules and the rRNA of ribosomes.

Processing the mRNA Transcript
Cap and Tail
After transcription, the mRNA transcript has both a cap and a tail added. The cap is added to the 5' end of the RNA molecule (the end first synthesized) and a tail of adenine nucleotides is attached to the 3' end of the mRNA transcript.

Both cap and tail help the mRNA attach to the ribosome for translation, and also inhibit enzyme degradation of the mRNA transcript.

Introns and Exons
The mRNA when it is released from RNA polymerase contains a mix of codable and non-coding RNA nucleotide sequences. The non-coding DNA segments are called introns (because they are intervening segments which interrupt the message) and do not code for amino acids.

Those regions that do code for amino acids are called exons (because they are expressed). Prior to using a mRNA transcript the introns must be removed, which is done during the RNA processing stage. This process is called RNA splicing, because the introns are cut out and the remaining exons get spliced together.

Special enzyme complexes are involved with the removal of introns. These complexes are called spiceosomes and are RNA-protein complexes. The removal of introns and splicing together of exons involves both protein enzymes and RNA catalysts. These RNA catalysts are called ribozymes, and are an important exception to the rule that all catalysts of living organisms are proteins.

Why Introns?
One area of interest is why so much of the DNA molecule contains introns. There is much research in this field. Some of the reasons might be:


Protein Synthesis – The Process of Translation
Once we have a final mRNA transcript, the mRNA is moved from the nucleus of the cell to the cytoplasm where is attaches to a small subunit of a ribosome and we are ready for the process of translation. Translation is where the information coded in DNA molecules is interpreted and translated to direct the actual synthesis of proteins. Translation also involves Initiation, Elongation and Termination.

Amino Acid Attachment
Prior to translation, one additional activity must occur: Amino acids must be attached to their appropriate tRNA molecules. The process of Amino acid attachment involves ATP and enzymes specific for each type of amino acid. Both the shape and charge of the tRNA molecules and the amino acids are important for the correct recognition and attachment

Although theoretically there should be 61 different tRNAs, one for each triplet code word other than the 3 stop triplets, there are about 45. As mentioned, the triplet code for DNA - amino acids is redundant. Often the third nucleotide is not crucial.

Initiation
The small rRNA subunit has a binding site for mRNA molecules during protein synthesis and the initiator tRNA. The larger rRNA subunit has three attachment sites for tRNA molecules, the P site, A site and E site. During protein synthesis the two subunits bind together.


Elongation

Termination

Summary of Transcription and Translation

Rate of polypeptide synthesis
A polypeptide is generally synthesized in about a minute. However, it is typical of mRNA to be working along many ribosomes at a time to direct the synthesis of many polypeptide molecules in sequence. As soon as the 5' cap of a mRNA leaves one ribosome it will attach to the small subunit of an adjacent ribosome to initiate protein synthesis at that ribosome. It is common for one mRNA to have many ribosomes associated at once. Such complexes are called polyribosomes.

Mutations – Mistakes in the DNA
Although the processes of DNA replication and RNA transcription are remarkable in their fidelity, sometimes mistakes are made. Each chromosome has a distinct pattern, size and shape. Each gene is a precise combination of DNA. Anything that affects the structure of a chromosome, the structure of DNA, the number of chromosomes typical for a species, or affects the ability of DNA to be transcribed accurately, is known as a mutation. We shall, at this time, discuss point mutations, changes that affect one or just a few nucleotides. Mistakes involving entire chromosomes are discussed along with chromosome behavior in inheritance.

While many times we stress that an alteration of the DNA produces harmful effects in the individual, it is by the act of mutation that many good variations also arise and can be passed on within populations. Without such variation, populations would not be able to respond to changing environments. Without such changes in the DNA over the millions of years of life on earth, we probably would not have the remarkable array of proteins, and hence, the remarkable array of life processes we have today.

The rate of mutation is highly variable, and depends in part of the ability of repair enzymes such as DNA polymerase and DNA ligase to fix mistakes. Some genes seem to mutate at much greater rates than others. The effect of a mutation is also highly variable. A mutation that does not harm the cell will be perpetuated in the cell line. Mutations that occur in gametes will be passed on to subsequent generations.

Mutations occur spontaneously as random events in cells. Mutations can also be induced. Substances that can promote mutations are known as mutagens.

Point Mutations
Base Substitutions
In a base substitution mutation, a single base pair is incorrectly matched, so that A will bond to C or G, rather than to T, for example. The DNA correcting enzymes may find the incorrectly matched pair, but might make the wrong correction, so that a different base pair results. This affects the "reading" of the gene, and may result in DNA instructions that can not be followed.

For example, the DNA for Hemoglobin normally codes for glutamic acid as the #6 amino acid. One hemoglobin mutation codes for valine in this position. The difference is Normal DNA code = CTC or CTT and Abnormal code = CAC or CAA. The result of this base substitution is the genetic disorder sickle cell anemia.

Insertions (Duplications)
If a new base pair is added to the DNA sequence being replicated, an insertion has occurred. The DNA instructions are now altered, and will be misread.


Deletions
In a deletion, a base pair is left out during DNA replication, and the DNA instructions cannot be read correctly.


Insertions and deletions will affect the reading of the entire gene past the point of change. (In theory, if an insertion or deletion involved complete triplet nucleotides, there would be just one amino acid affected, and the remaining codes could be read as usual.) In contrast, a base substitution may affect only a short sequence (one nucleotide triplet) of the DNA reading.

Reviewing the Effects of Mutations
As stated, a mutation may have no effect if a point mutation occurs at a place in the DNA coding that is redundant or if the mutation codes for an amino acid which will not alter the functioning of the protein, such as one in which the shape is not altered or the chemical nature of the protein is unchanged for its job.

Most commonly, the protein coded for by that gene will not be synthesized, or, if synthesized, can not function normally. For enzymes, this means that some biochemical activity in the affected cell will not occur. Depending on the specific activity not occurring, the mutation may or may not prove fatal to the cell, and in some cases, the organism.

In addition, if a mutated cell divides, any cells formed from that cell (DNA replication precedes cell reproduction) will perpetuate the DNA mutation. Keep in mind that a mutation may produce a change that improves the survival of the individual rather than being something that is harmful.

If a mutation occurs in the formation of gametes, and that gamete unites with another gamete, all cells of the new individual will have the mutation. The effect on the individual depends entirely on the specific mutation.

Again, mutations that are beneficial are an important source of genetic variation and are critical to the process of evolution

Regulating Genes
We have been discussing the structure of DNA and its role in protein synthesis. We have seen that DNA stores the information about how to assemble a protein, and that RNA molecules are used to transcribe and translate that information to direct the synthesis of specific proteins.

We have also discussed briefly a few effects of point mutations on gene expression.

We also know that each cell of an organism has exactly the same DNA, yet we have many different types of cells and tissues within an organism. Our DNA codes for about 30,000 proteins although not all of our DNA information is used in each cell, and not all information is used all of the time. We do not want to synthesize enzymes that are not needed, nor do we want to synthesize molecules in greater quantity than needed.

But how does a cell " "know" what DNA is needed and when? What controls gene activity? These are part of the subject of gene regulation. Biologists know that using genetic information in cells is a multi-step process that usually starts with transcription and “ends” with an enzyme catalyzing a particular chemical reaction on the cell. This process is sometimes called "information flow".

We shall try to provide some specific examples of how a gene is regulated as well as some general examples before leaving this section of biology.

Some of the answers to how genes are regulated are coming from work on recombinant DNA research; some from looking at genetics and, in particular, mutant strains of species. Unfortunately, less is known about the controls of gene activity and protein synthesis than we would like, although this is a very active area of research in developmental biology, the biology of aging, genetic diseases research, and cancer research.

Gene control is exerted chemically by molecules that interact with DNA, RNA and/or the polypeptide chains. Both hormones and enzymes have effects on gene expression.

Regulating Gene Expression - Prokaryotes
The early work on gene regulation was done with prokaryotes. It is easier to study activity in prokaryotes because they are less genetically complex, and absent a nucleus, the DNA is accessible to all components of the cell. Much of the research in gene regulation has been accomplished with Escherichia coli.

Recall that the typical gene codes for a polypeptide that is used to help the cell function in some way, or is a structural protein. A gene that codes for such proteins is a structural gene.

Other genes control how much of a polypeptide gets formed and when it gets formed. Such genes are regulatory genes.


The Operon of the Prokaryotic Cell
An active gene (or group of genes) includes the DNA that will be transcribed along with a promoter and operator. This complex is known as the operon and was described in 1961 by Francois Jacob and Jacques Monod. An operon has three parts plus an additional regulatory gene that activates or represses the operon.

Operon


Regulatory Gene

The Lactose Operon
We will look at the Lactose operon, described by Jacob and Monot in E. coli, for the gene that codes for the enzyme, lactase. In the Lactose operon, the substrate, allolactose (an isomer of lactose), attaches to a repressor protein that sits on the operator region of the gene.

In the absence of lactose, the repressor inhibits transcription by blocking RNA polymerase from attaching to the promoter. Once the lactose controller removes the repressor, the promoter is available and the genes that code for the three enzymes to digest lactose are transcribed.

When the enzymes are synthesized, the lactose is degraded, including the lactose molecules that are sattached to the repressor. When lactose is no longer available to bind to the repressor protein, the repressor shuts down the promoter (by sitting on the operator), which stops transcription.



Eukaryotic Gene Regulation
Like the prokaryotic gene, the eukaryotic gene has a number of regions, each important to transcription.

1. Control elements that consist of:


2. The codable gene including introns and exons
3. Termination Signals which end transcription


    Transcription Initiation Complex
    Transcription factors     bind to the enhancer (or silencer region of the gene if we are going to repress transcription instead) where activator proteins have attached. Hundreds of transcription factors have been identified in eukaryotes, and most likely are the direct control of transcription.

    The binding of activators to the enhancer results in bending the DNA molecule so that the activator proteins are brought more closely to the promoter region. This serves to attract more transcription factors to form a transcription initiation complex into which RNA polymerase can fit at the promoter region of the gene. When everything comes together, we get transcription.

    If a repressor has attached to the silencer region near the enhancers, activators are prevented from binding to the enhancers, and transcription is repressed.


    Having seen the level of complexity of the eukaryotic "gene", before we leave this subject, let's look at some of the general areas of control and mention a few examples of how gene activity is controlled.

    Some Examples of Eukaryotic Gene Controls



    Let's now look at some examples of eukaryotic gene regulation:

    DNA packing
    The DNA of eukaryotic cells is surrounded by a series of proteins which "package" the DNA on the chromosomes into bead-like units, called nucleosomes. These proteins may help to regulate gene activity by restricting access to specific genes.

    mRNA Stability
    The length of time a mRNA transcript can be read prior to degradation will affect the amount of final product, which will affect cell activity. Some mRNA lasts for weeks; some for hours.

    Hormone Activators
    Hormones are important chemical regulators for eukaryotes that can affect gene expression in their target cells. Some steroid hormones can act as activators for the promoter region of the gene, probably at the enhancer region of a gene.




    Product Inhibition
    A product can also serve to inhibit transcription. This has been shown with tryptophan, an amino acid. A high concentration of tryptophan stops the transcription of the enzyme which is needed to manufacture tryptophan.

    Condensed Chromosome Regions
    Most chromosomes have regions that are very tightly condensed. Transcription cannot take place in these regions.

    Chromosome Loops and Puffs and Copies
    Chromosome Inactivation
    Females have two X-chromosomes. In cells, one of them is deactivated and tightly condensed, the so-call Barr body, in which no transcription occurs. The choice of which X gets condensed for a given cell line appears to be random.

    Protein Modification
    Once a polypeptide is synthesized it can be altered by processing. Many metabolic proteins are non-functional until activated by other molecules. Hydrolytic enzymes, in particular, are synthesized in inactive forms. Membrane recognition proteins have additional molecules attached to them before they function. Regulation can also take place when proteins are moved from one part of a cell to another, or when exported from the cell.

    Protein Stability
    Proteins also vary in their "life span". Cells have mechanisms to degrade protein as well as other cell components. Cells can control the amount of protein by controlling the rate of degradation.

    Cancer and Gene Regulation
    Some cancers develop when the gene regulators are defective, and in all cancers, gene expression is defective.

    For example, when the normal cell division controls go awry, abnormal division results. One specific protein product of an oncogene (a cancer-promoting gene) that is important in cell division is the P53 tumor suppressor gene.

    P53 is a transcription factor for genes that keep a cell's DNA repaired and genes that delay the cell's rate of cell division so that there is time for DNA repair. If the cell is in bad shape, P53 activates cell suicide genes to prevent the harmful mutations from being passed on. Such cell death is called apoptosis. When p53 is defective or missing, cancers are more likely.


    P53 and Cancer


    How does one get cancer?
    Most believe that the onset of cancer is an accumulation of mutations rather than one single alteration. This correlates with the increase in many cancers with aging. Any number of things in our surroundings can activate oncogenes. Chemicals that do so are called carcinogens. Radiation and combustion products of tobacco are two of the most common carcinogens. Asbestos and some heavy metals in particulate form are also carcinogens. Many steroids in higher than normal concentrations are carcinogenic, and a high fat, low fiber diet is also suspected as being cancer promoting. Some viruses are also important in cancer formation.

    We still cannot answer the question of how one gets cancer or if or why one person will and another will not after exposure to the same potential carcinogens.


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