Chapter 7 – Complementary Experimental Tools  277

the expression of one or more genes is downregulated (which in molecular biology speaks

for “lowered”) or turned off entirely by the action of a small RNA molecule whose sequence

is complementary to a region of an mRNA molecule (which would ultimately be translated

to a specific peptide or protein). RNA silencing can be adapted by generating synthetic small

RNA sequences to specially and controllably regulate gene expression. Most known RNA

silencing effects operate through such RNA interference, using either microRNA or similar

small interfering RNA molecules, which operate via subtly different mechanisms but which

both ultimately result in the degradation of a targeted mRNA molecule.

Gene expression in prokaryotes can also be silenced using a recently developed technique

that utilizes clustered regularly interspaced short palindromic repeats (CRISPR, pronounced

“crisper,” Jinek et al., 2012). CRISPR-​associated genes naturally express proteins whose bio­

logical role is to catalyze the fragmentation of external foreign DNA and insert them into

these repeating CRISPR sequences on the host cell genome. When these small CRISPR DNA

inserts are transcribed into mRNA, they silence the expression of external DNA—​it is a

remarkable bacterial immune response against invading pathogens such as viruses. However,

the CRISPR are also found in several species that are used as model organisms including

C. elegans and zebrafish and can also be effective in human cells as a gene-​silencing tool.

CRISPR has enormous potential for revolutionizing the process of gene editing.

Transcription activator-​like effector nucleases (TALENs) can also be used to suppress

expression from specific to genes. TALENs are enzymes that could be encoded onto a plasmid

vector in a host cell. These can bind to a specific sequence of DNA and catalyze cutting of the

DNA at that point. The cell has complex enzyme systems to repair such a cut DNA molecule;

however, the repaired DNA is often not a perfect replica of the original that can result in a

nonfunctional protein expressed from this repaired DNA. Thus, although gene expression

remains, no functional protein results from it.

RNA silencing can also use upregulated (i.e., “increased”) gene expression, for example,

by silencing a gene that expresses a transcription factor (see Chapter 2) that would nor­

mally represses the expression of another gene. Another method to increase gene expres­

sion includes concatemerization of genes, that is, generating multiple sequential copies under

control of the same promoter (see Chapter 2).

Expression of genes in plasmids, especially those in bacteria, can be controlled through

inducer chemicals. These chemicals affect the ability of a transcription factor to bind to a

specific promoter of an operon. The operon is a cluster of genes on the same section of the

chromosome that are all under control of the same promoter, all of which get transcribed and

translated in the same continuous gene expression burst (see Chapter 2). The short nucleo­

tide base pair sequence of the promoter on the DNA acts as an initial binding site for RNA

polymerase and determines where transcription of an mRNA sequence translated from the

DNA begins. Insight into the operation of this system was made originally using studies of

the bacterial lac operon, and this system is also used today to control the gene expression of

recombinant DNA in plasmids.

Although some transcription factors act to recruit the RNA polymerase, and so result in

upregulation, most act as repressors through binding to the promoter that inhibits binding of

RNA polymerase, as is the case in the lac operon. The lac operon consists of three genes that

express enzymes involved in the internalization into the cell and metabolism of the disac­

charide lactose into the monosaccharides glucose and galactose. Decreases in the cell’s con­

centration of lactose result in reduced affinity of the repressor protein to the lacI gene that, in

turn, is responsible for generating the LacI protein repressor molecule that inhibits expression

of the operon genes and is by default normally switched “on” (note that the names of genes

are conventionally written in italics starting with a lowercase letter, while the corresponding

protein, which is ultimately generated from that gene following transcription and transla­

tion, is written in non-​italics using the same word but with the first letter in uppercase). This

prevents operon gene expression. This system is also regulated in the opposite direction by

a protein called CAP whose binding in the promoter region is inversely proportional to cel­

lular glucose concentration. Thus, there is negative feedback between gene expression and

the products of gene expression.