Bacterial Transcription: Induction and The Lac Operon

A continuation from previous transcription posts: Regulation and Attenuation and Initiation, Elongation, and Termination.

Induction

Gene induction is a phenomenon that is incredibly fast, taking only two to three minutes for a cellular response.  During induction, the actual enzyme levels in the cell rise, and inhibitors of protein synthesis prevent induction (providing further evidence that it is, in fact, protein synthesis that is necessary for induction).  The lac operon is the most commonly studied gene that uses induction for regulation.

During initial studies of the lac operon, there were two types of genes considered: structural and regulatory genes.  Structural genes are those that encode the actual metabolic enzymes; regulatory genes are involved in controlling the expression of the structural genes.  The lac operon was convenient for study because it had an observable phenotype (the production of a gene in the presence of glucose or lactose) and because mutants could be generated that had different phenotypes.  After mutagenizing bacteria, the researchers screened E.coli on plates with glucose and X-galactose.  Colonies of bacteria that were inducible turned white and did not express β-galactosidase, and regulatory gene mutants would be able to express β-galactosidase in the absence of lactose (and turn blue).  Mapping of the genes that were responsible for these phenotypes led to the identification of the o and i regions.  Mutations in either of these regions resulted in a constitutive phenotype. 

Further analysis of the lac operon and induction led to the creation of a model:  the i region codes for the inducer, which binds the lac operon DNA in the promoter region (identified via DNA-binding assays and footprint analysis).  Later structural studies identified the i protein contains an HTH motif, as well as IPTG-binding domains.  The i gene, called the repressor can bind the promoter region of the lac operon and prevent RNA polymerase from binding.  With the presence of glucose but not lactose, the lac repressor binds the operator sequence of the genome and it prevents RNA polymerase and its helper protein CAP (bound to cAMP).  CAP-cAMP induces a bend in the DNA, which allows RNA polymerase to bind, and CAP has its own binding site the DNA that helps to position the polymerase.  With the presence of lactose, the repressor binds to the lactose and no longer binds the operator.  Therefore, RNA polymerase can bind the promoter sequence and promote transcription of the lac genes.  However, in the absence of glucose, which is indicative of a high concentration of cAMP (low ATP), CAP binds cAMP and promotes stronger interaction of polymerase and the lac promoter.   

Bacterial Transcription: Control and Attenuation

A continuation from yesterday's post: Bacterial Transcription Initiation

Transcriptional Control and Attenuation

Like eukaryotes, bacteria must be able to control their gene activity.  Gene expression can be controlled at the transcriptional level in a few ways.  One is via alternative sigma factors (the protein that binds the -10 and -35 sites and positions RNA polymerase), which are involved in controlling expression of specialized operons.  For example, σ32 is involved in regulating heat shock genes, σ28 is for genes involved with motility and chemotaxis, σ54 is involved in nitrogen metabolism, and σ70 helps transcription of most genes. 

Another method of transcriptional control is via attenuation.  The most frequently cited example of attenuation is the trp operon, which has been studied extensively.  Initial observations indicated that when tryptophan was present for the bacteria, mRNA corresponding to the trp gene were short.  However, when tryptophan was limiting in the media, the mRNA transcript was longer.  If the researchers removed a short sequence of DNA, the mRNA was transcribed in full and genes were fully expressed.  This short sequence of DNA was termed the attenuator, or premature transcriptional stop. 

The trp operon codes for an mRNA with four different regions that can differentially bind to each other:  The second region can bind the first or third; the third can bind the second or fourth.  The first region contains two successive codons for tryptophan incorporation, which important for determining how the transcript is formed.  With high tryptophan, the ribosome moves along through the first region, without stopping at the successive tryptophan codons.  Because RNA polymerase has not had time to release the transcript before the ribosome translates through region one, causing regions three and four to bind, polymerase is forced off the mRNA and transcription is prematurely stopped.  This results in a shortened transcript when the cell has sufficient tryptophan.  In contrast, with low tryptophan levels in the cell, the ribosome will stall at the successive tryptophan codons because it is not able to quickly translate the mRNA.  This stalling allows for RNA polymerase to continue on its merry way and finish the full transcript because regions two and three (not three and four) bind.  

Bacterial Transcription: Initiation, Polymerization, Termination

After the last post, I thought I would be updating more often, but that just didn't happen.  Either way, here's a post about bacterial transcription!

Bacteria as an Experimental System

Bacteria are a common genetic system, but why exactly do we use these tiny organisms to perform so many experiments?  Because they’re easy to use, of course.  There area  number of benefits to using bacteria, including:

- Establishing basic biological principles

- Genetic manipulation

- Short generation time

- Simple growth conditions

-  High population density 

- Ability to witness rare events

- Ability to select for rare variants

Bacterial Transcription

Transcription is function that has been heavily studied in bacteria.  This first step in gene expression is facilitated by a single RNA polymerase of six subunits.  Eukaryotes, in contrast, have four polymerases (I, II, and III, as well as a mitochondrial or chloroplast polymerase).  In bacteria and eukaryotes, the initiation of transcription requires a complex of proteins to assemble and facilitate polymerization of RNA from DNA templates.  

Bacterial RNA polymerase consists of six subunits, as mentioned previously.  The β and β’ subunits perform the polymerization reaction.  Two α subunits regulate the frequency of initiation.  The ω subunit is involved in stability and assembly of the polymerase enzyme. 

RNA polymerase first binds to the promoter region of DNA using a σ factor, which binds two specific regions of the promoter. The core polymerase and σ factor slide along DNA until they come upon a promoter.  This closed complex that finds the promoter converts to an open complex (not requiring any ATP for this action), which favors the separation of the DNA strands.  At this point, RNA polymerase begins to make short RNA segments, as it “stutters” along the DNA.  Small RNA oligos are formed, and σ factor begins to dissociate from the polymerase enzyme.  At this point, elongation of RNA transcripts can occur, which results from a tightening of the clamp and the formation of the RNA exit channel.  During elongation, RNA polymerase adds nucleotides to the growing RNA transcript at a rate of about 50 per second.  With σ factor dissociated, the “rudder” of RNA polymerase pries the DNA/RNA hybrid apart. 

When RNA polymerase is to stop the transcription of a gene, it has a few options.  First, the gene itself may have an AT-rich region that forms secondary structures that inhibit transcription after they have been copied.  These hairpin secondary structures may open the exit channel, and due to the less stable A-U base-pairing between the DNA and RNA, the transcript is released.  Additionally, there is a rho-dependent transcriptional termination method.  Rho is a hexameric protein that wraps approximately 60 bp of mRNA.  Rho, once bound to mRNA, activates and uses its ATPase activity to move as an RNA-DNA helicase.  Once it has become active, rho begins to unwind the RNA from the DNA, and when it approaches the active site of RNA polymerase, the transcript is released from the DNA. 

Bacterial genes in general can be found  in either direction on the genome and are very rarely overlapping.  RNA polymerase recognizes a distinct region on the chromosome to initiate transcription.  To identify this site, DNA footprinting is used.

DNA footprinting:

1.      Bind RNA polymerase to a DNA strand of known length

2.      Randomly cleave the DNA by nuclease or chemical agents

3.      Remove RNA polymerase from the DNA

4.      Separate the DNA strands on an agarose gel.

By DNA footprinting, it was recognized that there is a specific region that is “empty” (the footprint) on the agarose gel corresponding to where RNA polymerase binds.  Genetic analysis has identified two regions where RNA polymerase binds: at -35 and -10 relative to the initiation site.  The consensus sequences are TTGACA and TATAAT, respectively.  

More about the Nucleus: Matrix, Envelope, Pores, Lamins

I needed that little break.  Exams have calmed temporarily and I have begun studying in earnest yet again.  Now I plan to take a slightly different approach with these updates by moving through the material chronologically, as it was taught.  Maybe using this method, I'll be able to refer back  and interlink posts more efficiently.

The Nuclear Matrix

As mentioned, DNA wrapped in nucleosomes loops and attaches to the nuclear matrix, but what exactly is the nuclear matrix?  Technically, the nuclear matrix consists of what is left after the DNA, lipid, and protein content of the nucleus has been cleared.  It consists of the nucleolus, the nuclear pore complex and lamina, and the internal nuclear matrix.  Many have argued that the nuclear matrix is simply an artifact of the extraction process.

The nucleolus in the nuclear matrix consists of ten chromosomes that converge to form a small compartment.  These ten chromosomes all contain genes for rRNA, and the nucleolus is where the rRNA is synthesized.  The ribosomal rRNA is synthesized by RNA pol I and III, and after synthesis, proteins are added to the rRNA while it is still in the nucleus.  Various subcompartments of the nucleolus have also been identified: the fibrillar center consists of the nucleolar organizer and the rDNA genes; the dense fibrillar (pars fibrosa) consists of the sites of transcription; and the granular (pars granulose) makes up the ribosome subunits. 

The Nuclear Envelope and Pore Complex Lamina

The nuclear envelope consists of a double membrane that connects to the ER on the outside and to the nuclear lamina (and heterochromatin) on the inside.  The lamin B receptor (LBR) can be found in the nuclear envelope, as it is an integral membrane protein.  Also contained in the envelope is the nuclear pore complex.  The NPC is from 50-to-150 nm in size, with about 5,000 found on the membrane per nucleus.  At the NPC is where the inner and outer membranes of the nuclear envelope come together, and the NPC is involved in communication between chromatin and events outside the nucleus. 

The nuclear pore complex can pass 500 macromolecules per second, with molecules less than 5000 Da passing freely; those macromolecules larger than 60 kDa barely enter.  The channel that allows passage of these molecules is only approximately 10 nm wide.  The complex itself is made of 1000 proteins called nucleoporins, forming an octomeric structure.  It functions to import nuclear proteins via nuclear localization sequences (NLSs), a protein sequence that signals nuclear import.  A protein with more NLSs is imported more frequently, though an NLS does not mediate retention of the protein.  The protein importin, made of α and β subunits, assists in protein import by binding the NLS.  The importin receptor recognizes the NLS and then migrates on FG repeats of the nucleoporins to dock and translocate through the pore.  The cargo (with its NLS) is then released to the nucleus. 

Export of mRNA and proteins from the nucleus is also important to the cell.  It has been discovered that the sequence of the RNA does not affect its ability to export from the nucleus where it originated.  However, it has also been discovered that different RNAs are exported via different pathways, which may be facilitated by RNA binding proteins that contain a nuclear export sequence (NES).  Exports are the receptors for these NESs and facilitate protein movement out of the nucleus.  Those proteins with both an NES and NLS are considered shuttle proteins. 

Nuclear import and export is heavily regulated in the cell.  Phosphorylation can affect nuclear import: direct phosphorylation o the NLS can inhibit transport.  Ran-GTP is another important factor that affects transport.  Ran-GTP in the nucleus binds to empty receptors and transport them to the cytoplasm, where they can reload with a piece of cargo.  Ran-GAPs in the cytoplasm facilitate the hydrolysis of GTP to GDP, which promotes translocation of Ran-GDP to the nucleus.  Once in the nucleus, Ran-GEFs promote the exchange of GDP for GTP.  The interaction of the different forms of Ran allow for the recycling of shuttling proteins, allowing them to move proteins in and out of the nucleus.

The Nuclear Lamina

As mentioned, the nuclear envelope surrounds the nucleus and connects to the lamina on its inner face.  The nuclear lamina itself is about 75 nm thick and is composed of proteins similar to intermediate filaments, called lamins.  Lamins come in three forms: A, B, and C.  Lamins A and C bind heterochromatin.  Lamin B binds the lamin B receptor (LBR), which is connected to the nuclear envelope as an integral membrane protein.  The lamin complexes also peripherally bind to the NPC.  Lamins maintain the nucleus in its spherical shape, and phosphorylation of A and C subunits solubilizes them during prophase, allowing dissolution of the nuclear lamina.  Lamin B remains attached to its receptor during prophase, however.  Importantly, mutations in these proteins can cause laminopathies because they are involved in nuclear organization, and mutations can result in inhibited DNA synthesis.

The Inner Nuclear Matrix

Study of the inner nuclear matrix has shown that the protein composition is cell-type specific.  Additionally, chromosomes are not positioned randomly in the nucleus.  This has been exported by FISH using whole-chromosome probes.  It appears that chromosomes occupy specific territories and, at least in yeast, they take the Rabl conformation, with the telomeres and centromeres directly interacting with the nuclear lamina.  Matrix proteins function in both replication and transcription, and mRNAs from active genes can be found in the matrix, as can newly replicated DNA.  Additionally, matrix proteins of cancerous cells are different from normal cells.

Chromatin Review Articles:

Campos, E. I. and D. Reinberg. 2009. Histones: Annotating chromatin. Annu. Rev. Genet. 43:559-599.

Clapier, C. R. and B. R. Cairns. 2009. The biology of chromatin remodeling complexes. Annu. Rev. Biochem. 73:273-304.

Kouzarides, T. 2007. Chromatin modifications and their function. Cell. 128:693-705.

Margueron, R. and D. Reinberg. 2010. Chromatin structure and the inheritance of epigenetic information. Nat. Rev. Genet. 11:285-296.

Rando, O. J. and H. Y. Chang. 2009. Genome-wide views of chromatin structure. Annu. Rev. Biochem. 78: 245-271. 

Oncogenesis Part 2: Genetic Instability, Colon Cancer, Transformation

Genetic Instability in Cancer Cells

A gene that receives a great deal of attention in cancer research is p53.  This protein acts as a tumor suppressor and is mutated in about 50% of all cancers.  Further, p53 is involved in a number of pathways, including apoptosis and genetic stability, so misregulation of the protein is a common factor in tumor cells.  Normally, very little p53 is present in cells, but it is induced during cellular stress.  When the cell experiences stress, p53 can induce apoptosis or cell cycle arrest by binding DNA and increasing p21 transcription, which acts as a CKI (see mitosis posts).  If p53 is lost, as it is in many cancers, the cell will replicate when it is not supposed to, and DNA accumulates a number of mutations (genetic instability).  Additionally, most cells will stop dividing when the telomeres shorten to a critical length, which is facilitated by p53.  When p53 is lost, even shortened telomeres don’t stop the cell from dividing, and genetic instability, again, is increased.  Some of these genetically unstable cells will upregulate telomerase, which will allow for continued proliferation. 

Colon Cancer Example

In normal colon cells, the APC protein inhibits cell cycle progression  by preventing Wnt from activating c-myc, which is required for progression from G₁ to S.  If APC becomes mutated, the cell can progress through mitosis unchecked.  Because APC acts as a tumor suppressor, an individual must have two alleles that become mutated.  Individuals with a germline mutation in APC have an increased risk of colon cancer.  Further, if Ras becomes unregulated, it can stimulate MAPK signaling, leading to uncontrolled proliferation.

Colon cancer progresses through a number of stages:

1. Normal epithelium
2. Hyperplastic epithelium (via loss of APC)
3. Early adenoma
4. Intermediate adenoma (via activation of K-Ras)
5. Late adenoma (via loss of Smad4 and other tumor suppressors)
6. Carcinoma (via loss of p53)
7. Metastasis

There exist a number of pathways that colon cells can become cancerous through the above stages.  The exact number of steps involved in malignant tumor progression is unknown, and the steps also vary based on type of tumor, though the general mechanism is similar.

Cell Senescence and Telomerase

In a study performed by Hayflick, cells that were explanted from tissue were shown to double roughly 60 times before entering senescence, a period when the telomeres are short, and the cells no longer proliferate.  Some cells are able to pass through the senescence stage and enter crisis, which lasts roughly 10-20 generations.  If a cell is able to pass through crisis and still undergo mitosis, it is considered immortal. 

Telomerase is the enzyme that can prevent cells from entering senescence.  When the catalytic subunit of telomerase (hTERT) is expressed, the telomeres are no longer degraded with each division.  With telomeres that are no longer shorted, there is no signal to p16^INK4A through pRb and p53 to enter senescence, and the cell continues to divide.  The expression of hTERT in HEK (hamster embryonic kidney) cells prevents the entry of the cells into senescence.

Growth Signaling and Transformation

In order for a cell to divide, it must receive a number of signals that indicate that the environment is appropriate for it to divide.  In addition to dividing, tumor cells must be able to grow in size.  The growth signaling pathway that has received the most attention has been that involving Ras.  Nonetheless, there are a number of pathways that feed into cell proliferation signals, and these are often the genes that are altered in cancerous cells. 

In culture, cells that have been transformed exhibit the ability to form foci.  Cells in tissue culture are usually inhibited when a confluent monolayer has been established.  Those cells that are able to grow on top of each other in an unregulated fashion are considered transformed.  In a 3T3 cell, a common cell type used for understanding oncogenes, those cells that form a focus and are transformed have a mutation in p16, leading to a loss of function (p16 is a CKI)

The 3T3 Transformation Assay

1. Transfect 3T3 cells with DNA from cancer cells
2. Allow the cells to form foci
3. Isolate DNA from foci and transform into new 3T3 cells
4. Isolate DNA from new foci and generate a phage library
5. Screen the phage library (with Alu probe) to identify human sequences

It is important to note that 3T3 cells are mouse cells, which facilitates the identification of human sequences using Alu elements (the mouse cells will not have these sequences).

Using the 3T3 transformation assay, the Ras onocogene was found.  The assay has also allowed for the identification of several other proto-oncogenes (genes that have the ability to become oncogenic).  Proto-oncogenes are usually activated via a gain-of-function mechanism, such as constitutive activity.  Approximately 100 oncogenes have been identified through the 3T3 assays and other methods.  Oncogenic collaboration is the cooperation between oncogenes to facilitate the faster formation of tumors. 

Proto-oncogenes can become oncogenes in several ways:

1. Point mutations conferring constitutive activity
2. Gene amplification leading to overexpression
3. Chromosomal translocations putting the proto-oncogene under the control of a different promoter
4. Chromosomal translocations that fuse two genes to make a chimeric protein with constitutive activity