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Summary - Molecular Basis of Gene Expression (5BBG0205)
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- Kings College London (KCL)
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Molecular basis of gene expressionNote the examination counts for 60% of the module mark and will be of 2 hours duration.
Section Format Percentage of module mark A Single best answer MCQs. 30% B Short answer
question (SAQ). 30%
• Explain how genes are expressed in prokaryotes and eukaryotes
• Discuss the many ways in which gene expression is regulated in eukaryotes
• Describe key molecular biology techniques for measuring gene expression
We measure gene expression, by quantifying the amount of a specific RNA or protein produced by a
cell
1. Reverse transcriptase PCR (RT-PCR)
2. Quantitative PCR (QPCR)
3. Northern blotting
4. Protein analysis (SDS-PAGE)
5. TRANSCRIPTOMICS
I. Gene expression microarrays
II. Next generation RNA-sequencing
Workshop topics
1. Promotor mapping using reporter genes
2. Applications of RNA interference (RNAi)
3. Post translational modification
4. Review of online quizzes
5. Real time PCR
6. Genomic technologies for measuring gene expression
, • Transcription - is the process of synthesis of RNA molecule by enzyme RNA polymerase using
DNA as a template, also a first step in gene expression.
• The DNA double helix must partially unwind for transcription to occur, this unwound region
is called a transcription bubble.
• Transcription is asymmetric – only one strand of the DNA is used as a template.
• The RNA is transcribed continuously.
• Each new RNA molecule is called a transcript.
• The region of DNA from which RNA is transcribed is called a transcription unit.
Transcription and processing of eukaryotic genes
The central dogma states that DNA, which contains the genetic code, is transcribed into RNA, and
RNA is then translated into proteins. This flow of information occurs in one direction, meaning that
information cannot be transferred from proteins back to DNA or RNA.
DNA is a double-stranded helix consisting of two complementary strands of nucleotides that run
antiparallel to each other. The strands are held together by hydrogen bonds (HBBs) between
complementary nucleotide bases,
Watson-Crick base pairs:
Complementary base pairs ensure that during DNA replication the order of bases in DNA can be
maintained by the DNA polymerase
Uracil in RNA
DNA and RNA interaction- transcription
RNA is single stranded, but can base-pair to form Watson-Crick base pairs: U-A, G-C
During transcription DNA-dependent RNA polymerase hybridises complimentary RNA bases and
strands from a single stand of DNA
,Transcription requires start and stop signals:
AUG – methionine – start codon for transcription
UAA, UAG, UGA – stop codons
Eukaryotes have three DNA-dependent RNA polymerases
Polymerase I – encodes for ribosomal RNA
Polymerase II – encodes for mRNA for proteins
Polymerase III – encodes for tRNA
Only ~3 % of all RNA produced is messenger RNA (mRNA) and codes for Protein
Start and END of protein coding genes are called promoter and poly(A) signal (PAS)
Not all genes are transcribed in every cell – Although every human cell has the same genotype,
differential gene expression creates different phenotypes
The stages of Pol II transcription and RNA processing
mRNA is modified during transcription to make it more stable and ready for translation
mRNA is processed prior to export from the nucleus to ribosomes in cytoplasm – Capping and
polyadenylation
- Transcription initiation depends on proteins binding DNA and Pol II.
- Capping: A 7-methylguanosine cap is added to the 5' end of the pre-mRNA. This cap helps
protect the mRNA from degradation and aids in its export from the nucleus. Cap that is
resistant to degradation by exonucleases – stable enough to be transported to cytoplasm and
translated. Capping essential for initiation of translation
, - Splicing: Introns, non-coding regions of the pre-mRNA, are removed by the spliceosome, a
large protein-RNA complex. The remaining exons are joined together to form the mature
mRNA. Splicing involves dynamic RNA base pairing and RNA-mediated catalysis
Alternative splicing – different isoforms of a gene can be created by splicing out exons
- 3’ end processing: Cleavage and polyadenylation
- Polyadenylation: An enzyme adds a string of adenine nucleotides (the poly(A) tail) to the 3'
end of the pre-mRNA. The poly(A) tail helps protect the mRNA from degradation and also
aids in its export from the nucleus.
Mammalian poly(A) signal is very conserved
Most genes have more than one poly(A) signal, distal poly(A) also carry UGUA
Untranslated regions have lots of elements that can be used for regulation and control of the RNA,
affects RNA stability
- How proteins “see” each other or DNA
- The major groove in DNA offers more electrostatic or hydrogen bond interactions than the
minor groove due to its wider and deeper structure, which exposes more nucleotide bases to
the solvent, providing more opportunities for protein-DNA interactions.
- However, the minor groove also plays a crucial role in protein-DNA recognition, particularly
for small molecules.
- Hydrophobic or van der Waals interactions/electrostatic interaction
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