Regulation of Gene Activity and Gene Mutations

15.1 Prokaryotic Regulation

Bacteria do not require the same enzymes all the time; they produce just those needed at the moment.

Francois Jacob and Jacques Monod (1961) proposed the operon model to explain regulation of gene expression in prokaryotes.

In the operon model, several genes code for an enzyme in the same metabolic pathway and are located in a sequence on a chromosome; expression of structural genes is controlled by the same regulatory genes.

An operon is a group of structural and regulatory genes that function as a single unit; it includes the following:

The trp Operon

Some operons in E. coli usually exist in the "on" rather than the "off" condition.

E. coli produces five enzymes as part of the anabolic pathway to synthesize the amino acid tryptophan.

If tryptophan is already present in medium, these enzymes are not needed and the operon is turned off .

The regulator codes for a repressor that usually is unable to attach to the operator.

The repressor has a binding site for tryptophan (if tryptophan is present, it binds to the repressor).

This changes the shape of the repressor that now binds to the operator.

The entire unit is called a repressible operon; tryptophan is the corepressor.

Repressible operons are involved in anabolic pathways that synthesize substances needed by cells.

 

trp operon

I = regulatory gene (codes for repressor protein)

P = promotor (RNA polymerase binding site)

 

O = operator (repressor protein binding site)

A-E = structural genes (code for tryptophan synthesis enzymes)

 

 

= RNA polymerase

 

The lac Operon

If E. coli is denied glucose and given lactose instead, it makes three enzymes to metabolize the lactose.

These three enzymes are encoded by three genes.

One gene codes for beta-galactosidase that breaks lactose to glucose and galactose.

A second gene codes for a permease that facilitates entry of lactose into the cell.

A third gene codes for enzyme transacetylase, which is an accessory in lactose metabolism.

The three genes are adjacent on a chromosome and under control of one promoter and one operator.

The regulator gene codes for a lac operon repressor protein that binds to the operator and prevents transcription of the three genes.

When E. coli is switched to medium containing an allolactose, this lactose binds to the repressor and the repressor undergoes a change in shape that prevents it from binding to the operator.

Because the repressor is unable to bind to the operator, the promoter is able to bind to RNA polymerase, which carries out transcription and produces the three enzymes.

An inducer is any substance (lactose in the case of the lac operon) that can bind to a particular repressor protein, preventing the repressor from binding to a particular operator; consequently, RNA polymerase can bind to the promoter and transcribe the structural genes.

 

 

lac operon

I = regulatory gene (codes for repressor protein)

 

P = promotor (RNA polymerase binding site)

 

O = operator (repressor protein binding site)

 

Z, Y, A = structural genes (code for lactose catabolism enzymes)

 

 

= RNA polymerase

 

Further Control of the lac Operon

Since E. coli prefers to break down glucose, how does E. coli know how to turn on when glucose is absent?

When glucose is absent, cyclic AMP (cAMP), which functions as a cellular alarm signal or alarmone, accumulates; cAMP has only one phosphate group and attaches to ribose at two locations.

CAP is a catabolite activator protein (CAP) in the cytoplasm.

When cAMP binds to CAP, the complex attaches to a CAP binding site next to the lac promoter.

When CAP binds to DNA, DNA bends, exposing the promoter to RNA polymerase.

Only then does RNA polymerase bind to the promoter; this allows expression of the lac operon structural genes.

When glucose is present, there is little cAMP in the cell.

CAP is inactive and the lactose operon does not function maximally.

CAP affects other operons when glucose is absent.

This encourages metabolism of lactose and provides a backup system for when glucose is absent.

Negative Versus Positive Control

Active repressors shut down the activity of an operon—this is negative control..

CAP is an example of positive control; when the molecule is active, it promotes the activity of the operon.

Use of both positive and negative controls allows the cell to fine-tune control of its metabolism.

If both glucose and lactose are present, the cell preferentially metabolizes glucose.

15.2 Eukaryotic Regulation

Different cells in the human body turn on different genes that code for different protein products.

Eukaryotes have four levels of regulatory mechanisms to control gene expression; two in the nucleus and two in the cytoplasm.

There are several levels of control that can modify the amount of gene product.

Chromatin structure: if genes are not accessible to RNA polymerase, they cannot be transcribed.

Transcriptional control in the nucleus determines which structural genes are transcribed and the rate of transcription; it includes transcription factors initiating transcription and transposons (DNA sequences that move between chromosomes and shut down genes).

Posttranscriptional control occurs in the nucleus after DNA is transcribed and preliminary mRNA forms.

Translational control occurs in cytoplasm after mRNA leaves the nucleus but before there is a protein product.

Posttranslational control occurs in the cytoplasm after protein synthesis.

Chromatin Structure

Eukaryotic DNA is in the form of chromatin, a stringy material associated with proteins.

The DNA is wound around a core of eight protein molecules ("beads on a string"); the proteins are called histones and each "bead" is called a nucleosome.

During interphase, some chromatin is highly compact, darkly stained, and genetically inactive heterochromatin.

The rest is diffuse, lightly colored euchromatin thought to be genetically active.

Barr bodies are an example of heterochromatin.

Since human males have only one X chromosome, it might be supposed that they produce half the gene product of a female with two X chromosomes.

However, females have in each nucleated cell a darkly staining Barr body, a condensed, inactive X chromosome.

Which X chromosome is condensed in each cell is determined by chance.

Thus, the body of heterozygous females is "mosaic"; half her cells express alleles on one X chromosome and half of her cells express the alleles on the other X chromosome.

Female gonads do not show Barr bodies; both X chromosomes are needed in development.

Only one active X chromosome in the female zygote means that lower X-coded gene products are normal.

Other examples of this mosaic effect include: ocular albinism, Duchenne muscular dystrophy, and female calico cat coat color.

Euchromatin activity is related to the extent nucleosomes are coiled and condensed.

A nucleosome is a bead-like unit made of a segment of DNA wound around a complex of histone proteins.

When DNA is transcribed, activators called remodeling proteins are able to push aside the histone proteins so transcription can begin.

Epigenetic inheritance is the term used to describe inheritance patterns that do not depend on the genes themselves.

Histones proteins have different chemical modifications in heterochromatin and euchromatin.

Methylation of DNA accounts for a phenomenon called genomic imprinting: gene expression is dependent on whether the chromosome carrying the gene is inherited from the mother or the father.

Epigenetic inheritance explains unusual inheritance patterns, and may have implications in growth, aging and cancer.

Transcriptional Control

Transcriptional control is the most critical level of genetic control.

Transcription is controlled by DNA-binding proteins called transcription factors.

Each cell contains many different types of transcription factors.

A group of transcription factors binds to a promoter adjacent to a gene; the complex attracts and binds RNA polymerase, but transcription may still not begin.

Transcription activators are often involved in controlling transcription in eukaryotes.

Transcription factors are always present in the cell and most likely they have to be activated in some way (e.g., regulatory pathways involving kinases or phosphatases) before they bind to DNA.

Transposons are specific DNA sequences that move with and between chromosomes.

Their movement may increase or decrease the expression of neighboring genes.

They are among the 40% of the human genome consisting of the same short sequence of DNA continuously repeated.

They are noncoding sequences that play regulatory functions, and could thus be considered part of epigenetic inheritance.

Posttranscriptional Control

Posttranscriptional control includes mRNA processing and the speed at which mRNA leaves the nucleus.

Messenger RNA molecules are processed before they leave the nucleus and enter the cytoplasm.

Differential excision of introns and splicing of mRNA can vary the type of mRNA that leaves nucleus.

The hypothalamus and thyroid glands produce calcitonin but the mRNA that leaves the nucleus is not the same in both types of cells.

Radioactive labeling shows they vary because of a difference in mRNA splicing.

Evidence of different patterns of mRNA splicing is found in cells that produce neurotransmitters, muscle regulatory proteins, and antibodies.

Speed of transport of mRNA from nucleus into cytoplasm affects the amount of gene product realized per unit time following transcription; there is a difference in the length of time it takes various mRNA molecules to pass through nuclear pores.

Translational Control

Translational control begins when the processed mRNA molecule reaches the cytoplasm and before there is a protein product.

The longer an active mRNA molecule remains in the cytoplasm, the more product is produced.

Mature red blood cells eject their nucleus but synthesize hemoglobin for several months; the mRNAs must persist during this time.

Ribonucleases are enzymes associated with ribosomes that degrade mRNA.

Mature mRNA has non-coding segments at 3' cap and 5' poly-A tail ends; differences in these segments influence how long the mRNA avoids being degraded.

MicroRNAs are small, processed pieces of intron; after microRNAs are degraded, they combine with protein and the complex binds to mRNAs, destroying them.

Posttranslational Control

Posttranslational control begins once a protein has been synthesized and has become active.

Some proteins are not active after synthesis; the polypeptide product has to undergo additional changes before it is biologically functional.

Bovine proinsulin, for example, is inactive when first produced; a single long polypeptide folds into a three-dimensional structure, a sequence of 30 amino acids is removed from the middle, and the two polypeptide chains are bonded together by disulfide bonds resulting in an active protein.

Many proteins are short-lived in cells and degraded or destroyed so they are no longer active.

Giant protein complexes called proteasomes carry out this task.

One example is the cyclins that control the cell cycle; they are only temporarily present.

15.3 Genetic Mutations

A genetic mutation is a permanent change in the sequence of bases in DNA; mutations range from having no effect to total inactivity.

Effect of Mutations on Protein Activity

Point mutations change a single nucleotide and therefore change a single specific codon.

The effect of the point mutation depends on the specific base change in the codon.

Changes to codons that code for the same amino acid have no effect; e.g., UAU to UAC both code for tyrosine.

A change from UAC to UAG (a stop codon) results in a shorter protein, and a change from UAC to CAC incorporates histidine instead of tyrosine.

Sickle cell disease results from a single base change in DNA where the beta-chain of hemoglobin contains valine instead of glutamate at one location and the resulting distorted hemoglobin causes red blood cells to clog vessels and die off sooner.

Frameshift Mutations

The reading frame depends on the sequence of codons from the starting point: THE CAT ATE THE RAT.

If, for example, C is deleted, the reading frame is shifted: THE ATA TET HER AT.

Frameshift mutations occur when one or more nucleotides are inserted or deleted from DNA.

The result of a frameshift mutation is a new sequence of codons and nonfunctional proteins.

Nonfunctional Proteins

A single nonfunctioning protein can cause dramatic effects.

The human transposon Alu is responsible for hemophilia when it places a premature stop codon in the gene for clotting factor IX.

PKU results when a person cannot convert phenylalanine to tyrosine; phenylalanine builds up in the system, leading to mental retardation.

A faulty code for an enzyme in the same pathway results in an albino individual.

Cystic fibrosis is due to inheriting a faulty code for a chloride transport protein in the plasma membrane.

Androgen insensitivity is due to a faulty receptor for male sex hormones; body cells cannot respond to testosterone and the individual develops as a female (even though all of the body cells are XY).

Carcinogenesis

The development of cancer involves a series of various types of mutations.

Tumor-suppressor genes normally act as brakes on cell division when it begins to occur abnormally.

When proto-oncogenes mutate, they become oncogenes.

Tumor-suppressor genes and proto-oncogenes often code for transcription factors or proteins that control transcription factors.

P53, a major tumor-suppressor gene, is more frequently mutated in human cancers than any other known gene.

The p53 protein acts as a transcription factor to turn on the expression of genes whose products are cell cycle inhibitors.

The p53 can also stimulate apoptosis (programmed cell death).

Other proto-oncogenes code for Ras proteins, which are needed for normal cell growth and DNA synthesis.

Inheritance of Cancer

Genes called BRCA1and BRCA2 are tumor-suppressor genes that behave as outosomal recessive alleles.

A mutated RB allele causes cancer of the eye; it takes another mutated allele to increase the chance of cancer.

The inherited RET gene predisposes a person to thyroid cancer.

 

Cause of Mutations

Some mutations are spontaneous, others are due to environmental mutagens.

Mutations due to replication errors are very rare.

DNA polymerase constantly proofreads new DNA against the old, and repairs any irregularities, thereby reducing mistakes to one out of every one billion nucleotide pairs replicated.

Environmental mutagens are environmental substances that increase the chances of mutation.

Common mutagens are radiation and organic chemicals.

Carcinogens are mutagens that increase the chances of cancer.

Tobacco smoke contains a number of known carcinogenic chemicals.

X rays and gamma rays are ionizing radiation that creates free radicals, ionized atoms with unpaired electrons.

Ultraviolet (UV) radiation is easily absorbed by pyrimidines in DNA.