BCHM 416

Altering the amount of enzyme

• Another long duration method of controlling the activity of an enzyme is to
alter the activity of an enzyme. Thus
• The amount of a particular enzyme depends on both its rate of synthesis
and its rate of degradation. The level of most enzymes is adjusted primarily
by changing the rate of transcription of the genes encoding them.
• The amount of enzymes (proteins) in cells is regulated at the levels of
transcription, post-transcription, translation, post-translation, and
degradation.
 Regulation of Enzyme activity via gene expression
• Transcriptional Regulation:
• Gene Expression: The amount of enzyme produced is controlled at the gene transcription
level. Transcription factors and other regulatory proteins bind to promoter regions of
genes, enhancing or repressing transcription.
• Inducers and Repressors: In prokaryotes, operon systems (e.g., lac operon) use inducers
and repressors to regulate enzyme synthesis in response to environmental changes. In
eukaryotes, more complex regulatory networks involving enhancers, silencers, and
transcription factors exist.
• Post-Transcriptional Regulation:
• mRNA Stability: The stability and degradation rate of mRNA transcripts affect how much
enzyme is synthesized. Regulatory elements in the mRNA sequence, such as AU-rich
elements, influence mRNA stability.
• Alternative Splicing: This process generates different mRNA variants from a single gene,
leading to the production of different enzyme isoforms with distinct activities.
 Regulation of Enzyme activity via gene expression
• Translational Regulation:
• Ribosome Binding: The initiation and rate of translation can be regulated by
factors that influence ribosome binding to mRNA. For instance, iron levels
regulate the translation of ferritin mRNA by affecting the binding of iron-
regulatory proteins.
• Post-Translational Modifications:
• Protein Processing: Newly synthesized enzymes often undergo modifications
such as glycosylation, phosphorylation, and proteolytic cleavage, which are
essential for their stability, localization, and activity.
• Degradation:
• Proteasome and Lysosome Pathways: Enzymes are targeted for degradation
by the ubiquitin-proteasome system or lysosomal pathways. Ubiquitination
marks proteins for degradation, ensuring that enzyme levels are tightly
controlled.
 Regulation via transcription
• A gene is expressed when in is transcribed into mRNA and (for most genes)
translated into protein.
• Genes can be constitutive or inducible (regulated) in terms of expression.
• Regulated genes possess other sequences in the genome referred to as
“regulatory sites” often located some distance from the gene.
• Regulatory sites and usually binding sites for specific DNA-binding
proteins. Such binding may either stimulate or repress gene expression.
• The basic mechanism by which transcription is regulated depends on highly
specific interactions between transcription regulating proteins and
regulatory sequences on DNA.
 Lac-operon
• The “lac operon” is an inducible catabolic operon of E. coli.
• It carries three structural genes: ‘Z’, ‘Y’ and ‘A’. that code for β-
galactosidase, galactoside permease and thiogalactoside transacetylase
respectively. (polycistronic mRNA)
• It possesses the promoter (P) region; a short sequence of DNA flanking
the origin of transcription.
• The promoter can be controlled by one or more regulatory proteins that
responds to chemical signals (environmental stimuli) and
• The operator (O) region; which is also a short segment of DNA adjacent
to the promoter and acts as a control element to a regulator protein.
 Mechanism of lac operon regulation
• In the absence of lactose in E. coli, the cells do not need to express
these genes;
• the regulatory protein specifically binds to the operator gene (O).
• This prevents the binding of the enzyme RNA polymerase to the
promoter site (P), thereby blocking the transcription of structural
genes (Z, Y and A), hence reduces the rate of transcription initiation
• The repressor molecule acts as a negative regulator of gene
expression.
 Mechanism of operon regulation
• In the presence of lactose in E. coli,
• An inducer (signal) molecule binds to a specific site on the Lac
repressor, causing a conformational change that results in
dissociation of the repressor from the operator.
• the repressor protein dissociates from the operator gene (O).
• This enhances the binding of the enzyme RNA polymerase to the
promoter site (P), thereby inducing the transcription of structural
genes (Z, Y and A), hence increases the rate of transcription
initiation
• The repressor molecule acts as a positive regulator of gene
expression.
Post transcriptional processes
• Transcript has to undergo a series of processing steps before
they are exported from the nucleus to translation machinery:
• the mRNA acquires a m7G cap structure at the 5’ terminus,
• introns are spliced out from the pre-mRNA, and
• a specialized 3’ end of the mRNA is generated, usually by
polyadenylation.
• These maturation steps are cotranscriptional and can
influence each other’s activities
Post-Transcriptional Regulation
• mRNA Stability: The stability and degradation rate of transcripts
(mRNA) affect how much enzyme is synthesized. Regulatory elements
in the mRNA sequence, such as AU-rich elements, influence mRNA
stability.
• Alternative Splicing: This process generates different mRNA variants
from a single gene, leading to the production of different enzyme
isoforms with distinct activities.
Regulation of gene expression via mRNA stability
• This can be achieved through several mechanisms that affect how long an
mRNA molecule remains intact and available for translation into protein.
• mRNA sequence elements: the 5’ cap and 3’ poly(A) tail protect mRNA from
degradation. Thus enzymes that remove these structures can trigger rapid mRNA
degradation.
• AU-rich elements (AREs) these are found in the 3’ untranslated region (UTR) of many
mRNAs. AREs can enhance mRNA degradation when bound by specific proteins.
• RNA-binding proteins (RBPs) proteins that bind to mRNA and protect/promote
degradation, e.g. HuR binds to AREs and stabilizes mRNA while Tristetraprolin (TTP)
binds to AREs and recruits degradation machinery.
• MicroRNAs (miRNAs) this are small non coding RNAs that bind to complementary
sequences in the 3’ UTR of target mRNA destined for degradation or translational
repression, thus reducing the amount of product.
Regulation of gene expression via alternative splicing
• During mRNA processing, alternative splicing patterns can change, thus
production of variant mRNA transcripts and different protein isoforms at
different developmental stages.
• Production of different isoform: isoforms with different functions, cellular
localizations or interactions.
• Regulation of mRNA stability and translation: splicing can influence the
inclusion/exclusion of regulatory sequences in the mRNA transcript, such as binding
sites for RBPs or miRNAs. This can affect mRNA stability and its translation efficiency.
• Functional modulation of proteins: alternative splicing can affect the functional
domains of proteins; enzymatic activity, binding properties and interactions with other
proteins.
• Control of protein localization: splicing can influence the inclusion of localization
signals within proteins determining their destination.