Slide 1

Gene Expression 2
Regulation
Ericka Ann Lawson, Ph.D.
ealawson@swbell.net

Figures obtained from: Wikipedia https://en.wikipedia.org/wiki/Regulation_of_gene_expression ,

Wikipedia
Slide 2

Objectives
1. Describe the structure of a typical protein-coding human gene, and the functional significance of its components.
2. Name two kinds of epigenetic modifications and their consequences for transcription.
3. Describe how splicing occurs, alternative splicing, and how splice site mutations can cause disease.
4. Explain the functions of promoter, enhancers, silencers, and transcription factors.
5. List the kinds of repetitive sequences in the human genome, their structures and their origins.
6. Explain how mobile sequences can cause problems.
7. Explain the origins of pseudogenes and processed pseudogenes.
8. Given a mutation that has occurred in a DNA sequence, determine the effect of that mutation on gene expression.
9. List the ATP- and GTP-dependent processes of protein synthesis.
10. Describe the roles of ribosomal subunits, ribosomal RNA, and the initiation and elongation factors in protein synthesis.
11. Describe the steps involved with the initiation, elongation and termination of translation.
12. Specify the mechanisms by which antibiotics interfere with translation.
13. Describe how ricin, polio virus and diphtheria toxin interfere with translation.
Slide 3

Antibiotics differentiate between prokaryotic

and Eukaryotic ribosomes.
Slide 4

Energy usage is dependent on availability of

GTP and ATP
• These enzymes use ATP or GTP for each cycle of action.
• Histone acetylase
• Topisomerases
• Helicases
• DNA pol beta in proofreading in replication
• Ligase
• Capping enzyme
• Spliceosomes
• nucleotidyltransferase
• Aminoacyl tRNA synthase
• Translation initiation factor
• EF Tu in placing charged tRNA in A site of ribosome
• eEF2 in translation translocation
• Translation Release factor
Slide 5

Regulation of DNA packing

• Acetylation/deacetylation of H4
• Methylation of Cytosine
• Binding of replication or transcription factors
Slide 6

Regulation of Replication
• Pre-replication complex (pre-RC) assembly and activation by CDK and
DDK phosphorylation *G1 CHECKPOINT*
• Loss of Cdc 6 and gain of Cdc45 to pre-RC to form initiation complex
• GINS complex binding to origins of replication (also required for
maintenance of the replication fork)
• PCNA/DNA Pol beta (required for both synthesis and base excision
repair for editing)
• Replication Factor C (RPC) loads PCNA onto DNA at origin and for
each Okazaki fragment.
• Availability of ATP, dATP, dCTP, dGTP and dTTP
Slide 7

Incorporation of dideoxynucleotides
(ddNTP) into DNA results in the loss of the
3’OH and the immediate termination of
polymerization.

Antiviral drug Acyclovir (Zovirax) is Dideoxyguanosine,

(ddGTP). Acyclovir is used in the treatment of some viral
infections because it terminates replication. Herpes
Simplex and Varicella Zoster

Use of dideoxynucleotide termination of Acyclovir in conjunction with AZT (Zidovudine) are used to
replication is the basis of the Sanger treat HIV because together they inhibit the reverse
sequencing method used to sequence
the human genome. transcription and the replication of the virus
https://upload.wikimedia.org/wikipedia/commons/b/b2/Sanger-sequencing.svg

1) Dideoxynucleotides are used as a pharmaceutical regulator of replication. Typically, these

are used to treat herpes and chicken pox.
2) In HIV treatment as well as for some other viruses, AZT is added to the treatment because it
inhibits the reverse transcriptase by modification. AZT can also inhibit replication, but the
levels needed to fully inhibit replication in humans is above the therapeutic range.
3) Incorporation of ddNTP and AZT results in strand termination due to the loss of a 3’OH after
it is incorporated.
4) ddNTPs are also used in DNA sequencing.
Slide 8

Pharmaceuticals that inhibit replication

directly or via substrate depletion
• Inhibitors of Polymerases • Inhibitors of dTTP production
• Cytarabine (Ara-C®) • 5-Fluorouracil (5-FU)
• Floxuridine • 6-mercaptopurine (6-MP)
• Fludarabine • Capecitabine (Xeloda®)
• Gemcitabine (Gemzar®) • Floxuridine
• Fludarabine
• Hydroxycarbamide
• Methotrexate
• Pemetrexed (Alimta®)
Slide 9

Pharmaceutical agents can be used to preferentially inhibit nucleic acid synthesis in bacteria
while not affecting the Eukaryotic Machinery for either replication or transcription.
Slide 10

Regulation of Transcription
• Availability of ATP, CTP, GTP AND UTP
• Exposed region of DNA containing the TATA box and RNA pol binding
motif
• Tissue specific enhancers or repressors
• Cell cycle specific enhancers or repressors
• Metabolic state specific enhancers or repressors
Slide 11

Enhancer induced looping of the gene

Transcription factor binding sites

• upstream of the RNA pol binding site
• Up to 10 kb upstream
• May have multiple enhancers for the same
gene
• Allows for tissue specific regulation
• Environmental regulation
• Developmental regulation
• Cell cycle dependent regulation
• Circadian regulation

By Jon Cheff - Own work, CC BY-SA 4.0,

https://commons.wikimedia.org/w/index.php?curid=40467125

1) Gene structure reminder

2) Repressors typically bind near the RNA entry site or the transcriptional start site and block
transcription by physical obstruction.
3) Enhancers can bind near but typically bind at a distance, then bend DNA as it associates with
transcription factors that recruit RNA polymerase to its binding site.
4) The availability of specific enhancers regulate transcription to achieve the needs of the cell
or tissue.
Slide 12

External signals regulate gene expression

Hormonal regulation is typically up regulation

of transcription either by direct binding of the
hormone-receptor complex or a second
messenger in that hormonal cascade. Ex.,
Vitamin D3-receptor complex up regulate
calbindin expression to illicit increased uptake
of calcium by the intestines

Some hormonal signals down regulate

transcription and do so in a similar fashion to
up regulation of other genes.

https://upload.wikimedia.org/wikipedia/commons/e/e6/Regulation_of_gene_expression_by_s
teroid_hormone_receptor.svg

1) Second messenger pathways alter the availability of specific enhancers and repressors.
2) Some hormone regulation of transcription is via direct DNA binding of the hormone. Steroid
hormone receptors are a good example.
Slide 13

Regulation of Transcript Maturation

• C-terminus of the RNA pol II phosphorylation state
• Splice sites containing canonical sequence motif and folding
5’GU-------A--py py py ---AG3’
• Tissue specific RNA binding proteins
• Cell cycle specific RNA binding proteins
• Metabolic state specific RNA binding proteins
Slide 14

RNA folds into many

shapes

dsRNA is helical

Frequent bulges or loops

result in more complex
shapes

RNA folding is required for

self splicing of tRNA and
rRNA https://publications.nigms.nih.gov/thenewgenetics/chapter2.html
Slide 15

Regulation of Translation
• 5’ cap (entry point for small subunit of ribosome)
• Availability of ATP and GTP
• Initiation factors
• Elongation factors
• Availability of Aminoacyl tRNA
• Phosphorylation of translation factors
Slide 16

Antibiotics differentiate between prokaryotic

and Eukaryotic ribosomes.

1) As for nucleic acid synthesis there are specific pharmaceutical inhibitors of protein synthesis.
2) The antibiotics have no effect on the Eukaryotic Ribosome.
Slide 17

Polio Virus
• Polio virus is a ssRNA virus
• The virus contains the + strand (coding) and is used for translation and
replication of the viral genome.
• 5’end of the vRNA is highly structured and recruits hosts ribosome
assembly for rapid translation of the genome. (IRES)

https://upload.wikimedia.org/wikipedia/commons/6/65/Poliovirus_genome.png
Slide 18

Ricin
• Lecithin from Castor beans
• Lethal in humans at 1 mg/kg body mass
• Breaks the glycosidic bond of A4324 in the 28S rRNA (large subunit)
• A4324 and its surrounding sequence is required for binding of
elongation factors.
Slide 19

Diphtheria Toxin
• Secreted dimer from Corynebacterium diphtheriae or as a monomer
from Pseudomonas aeruginosa)
• The A subunit is processed in an endosome and released into the
cytosol.
• Elongation factor 2 eEF-2
• Normally uses GTP as the energy source for peptide bond formation
• ADP-ribosylated by the cTerminal domain of the A subunit
• After ADP ribosylation the eEF2 cannot catalyze GTP …thus peptide bond
formation is prevented.
Slide 20

Recap of global concepts

• All stages of nucleic acid metabolism undergo regulation
• Common methods of regulation
• Availability of substrates, enhancers, repressors and associated factors
• Protein modifications via acetylation, methylation, phosphorylation and ADP
ribosylation
• competitive and non-competitive binding of pharmaceuticals
• Physical obstruction of activity

THIS SLIDE IS NOT A COMPLETE REVIEW. Please study all slides and additional reading and
resources to fully prepare for the exam. You will be asked about the specifics of these concepts
and be given hypothetical scenarios to solve that will require you to understand this material at
a much deeper level than this slide.