Transcript Recombinant DNA Technology

Eukaryotic Gene Expression
Managing the Complexities of
Controlling Eukaryotic Genes
Prokaryotes vs. Eukaryotes
Prokaryotes
Eukaryotes
Closely related genes are
clustered together
Related genes are located on
different chromosomes
Largely transcriptional
control
Significant transcriptional
control; other levels of control
possible
Trans-acting sequencespecific DNA binding
proteins
Proximal Cis-acting
sequences
Larger number of larger-sized
genes
Trans-acting sequencespecific DNA binding proteins
Cis-acting sequences can be
located at significant
distances
Control Points for
Gene Expression in Eukaryotes
DNA
Transcriptional Control
Post-Transcriptional Control
Translational Control
transcription
RNA
translation
Protein
Post-Translational Control
Levels of Eukaryotic Chromatin Structure
First level of chromatin coiling
Nucleosome = DNA + histone proteins
Variations in chromatin condensation affect gene activity.
Transcriptional Regulation:
Effects of Chromatin Structure
Decompaction of chromatin:
• Transcription factors unwind
nucleosomes in the area where
transcription will begin, creating
DNAse I hypersensitive sites
• RNA polymerase unwinds more
nucleosomes as transcription
proceeds
Transcriptional Regulation:
Effects of Chromatin Structure
Acetylation of histone proteins (adding -CH3CO)
reduces DNA-histone interaction, permitting
transcription factors to bind.
Transcriptional Regulation:
Effects of Chromatin Structure
DNA Methylation
• DNA Methylation (adding -CH3) can occur on
cytosines at CpG groupings near transcription
start sites
•Inactive genes have methylated cytosines
•Active genes have demethylated cytosines
• Acetylation of histones is associated with
cytosine demethylation
Transcriptional Regulation: Control of Initiation
•Transcriptional Activator Proteins assist in the
formation or action of the basal transcription
apparatus
Transcriptional Regulation: Control of Initiation
• Transcriptional Activator Proteins bind to
Enhancer sequences that increase
transcription
– Enhancers can influence
promoters at distances
of 50 kb or greater
due to DNA looping
mechanism
– Insulators control the direction of enhancer action
Transcriptional Regulation: Control of Initiation
• Transcriptional Repressor Proteins have three
possible modes of action
– compete with activators for DNA binding
sites
– bind to sites near activator site and inhibit
activator contact with basal transcription
apparatus
– interfere with assembly of basal transcription
apparatus
Post-Transcriptional Regulation:
Alternative RNA Splicing
Post-Transcriptional Regulation:
RNA Editing
Base substitution
= after transcription
Translational Regulation:
RNA Stability
• Degradation of mRNA can occur from the
5’ or 3’ end
• Stability of mRNA depends on
– 5’ cap
– 3’ poly-A tail
– 5’ and 3’ UTRs: serve as binding sites for
regulatory factors
– Coding region
• Example: Hormone prolactin increases the
longevity of casein mRNA coding for milk
protein in lactating mammals
Translational Regulation
• Masking of mRNAs
– Many species store mRNAs in the cytoplasm
of the egg. These mRNAs are inactive due
to masking by proteins. Fertilization of the
egg initiates unmasking and translation of
these mRNAs.
• Availability of specific tRNAs
– In the embryonic development of a
hornworm, an mRNA is present from day 1
but a specific tRNA needed for its translation
is not produced until day 6.
Translational Regulation:
RNA Silencing
Small
interfering
RNAs
microRNAs
RNAinduced
silencing
complex
Post-Translational Modification:
Phosphorylation
Addition or removal of a
phosphate group is a
common way to change
protein activity.
Post-Translational Modification:
Peptide cleavage
Proteins that have an inactive form after
synthesis are activated by removal of a
small number of amino acids.
Prothrombin
Cleavage
Thrombin
Activation of blood clotting
factors by cleavage
Fibrinogen
Cleavage
Fibrin
Fibrin
polymer
(blood clot)