Prokaryotic and Eukaryotic Genome Flashcards
Explain why the end replication problem occurs
-During DNA replication, DNA polymerase requires a free 3’OH end of a pre-existing strand to add free nucleotides
-An RNA primer is synthesised to provide this free 3’OH end for the addition of free nucleotides
-However, this RNA primer at the end of the DNA strand is removed and cannot be replaced by nucleotides, creating a 3’ overhang at end of chromosome
Explain how DNA methylation downregulates or silences gene expression at the chromatin level
DNA methylation * involves addition of a methyl group to selected cytosine (C) nucleotides located in the CG sequences.
-DNA methylation usually prevents transcription by:
(a) blocking binding of general transcription factors and hence, preventing assembly of transcription initiation complex at promoter
(b) recruiting DNA-binding proteins (i.e. transcriptional repressors, histone deacetylases and repressive chromatin remodeling complexes) to the methylated DNA to condense chromatin
-results in gene silencing/no gene expression
Describe how histone modification (e.g. acetylation and deacetylation) regulates gene expression at the chromatin level.
Acetylation and deacetylation of histones allows chromatin to decondense and condense, alternating between loose and tightly condensed states.
Acetylation of histones is catalysed by histone acetyl transferase
-addition of acetyl groups (-COCH3) to lysine residues removes positive charges on histones, decreasing the electrostatic interactions between the negatively-charged DNA and the histones.
Tight binding between DNA and histones is loosened, making promoter region more accessible to RNA polymerase and general transcription factors.
Acetylation works in concert with chromatin remodeling complex, allowing formation of the transcription initiatin complex
Deacetylation of histones is catalysed by histone deacetylase -> removal of the acetyl groups, restores a tighter interaction between DNA and histones inhibiting transcription
Compare gene expressions in euchromatin and heterochromatin, with reference to chromatin remodelling complexes.
(a) Heterochromatin = highly compacted DNA where DNA winds more tightly around histones.
-formation of heterochromatin results in silencing of genes/inactive gene expression as it limits access of RNA polymerase and general transcription factors to promoters of genes and thus prevents formation of the transcription initiation complex.
(b) Euchromatin = less compacted DNA where DNA winds less tightly around histones
-formation of euchromatin allows access of RNA polymerase and general transcription factors to promoters of genes hence allowing the formation of the transcription initiation complex.
Chromatin remodeling comnplex are protein complexes that alter structure of nucleosomes temporarily:
results in DNA being less/more tightly bound to histones
State the function of the promoter.
- located just upstream of the transcription start site of a gene
-Function as recognition site for the binding of general transcription factors and RNA polymerase to start/initiate transcription. - has critical elements
e.g.1. TATA box at -25 site
-important in determining precise location of the transcription start site
e.g. 2. CAAT and GC boxes:
-improve efficiency of promoter by helping to recruit general transcription factors and RNA polymerase to the promoter.
State function of the enhancer sequence.
-Enhancer sequences allow the binding of specific transcription factors called activators -> increases the frequency of transcription of genes they control -> gene activation
-Enhancers are positive regulatory elements involved in the upregulation of transcription as they promote the assembly of the transcription initiation complex via their interaction with activators
Explain how activators bind to enhancers to increase frequency of transcription
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Promoting assembly of transcription initiation complex
-Upon binding of activators to enhancers, spacer DNA bends
-Bending of spacer DNA allows direct interaction of activators with RNA polymerase and/or general transcription factors at the promoter, promoting the assembly of transcription initiatin complex. -
Increaseing accessibility to promoter DNA
-Bound activator may recruit histone acetyl transferase and chromatin remodeling complex to decondense chromatin
-Allows greater accesbility of general transcription factors and RNA polymerase to the promoter.
State the function of silencer sequence.
Silencer sequences allow binding of specific transcription factors called repressors which represses/prevents/decreases the frequency of transcription of the genes they control -> gene silencing/gene repression
-Silencers are negative regulatory elements involved in the downregulation of transcription as they prevent the assembly of the transcription initiation complex via their interaction with repressors
Explain how repressors bind to silencers to decrease frequency of transcription.
- Interfering with action of activator
A. Competitive binding
-Enhancer region overlaps with silencer region. Binding of repressor prevents binding of activator.
B. Masking activation surface
-Repressor binds to activator to prevent it from interacting with general transcription factors
C. Direct interaction with general transcription factors
-Repressor interacts with general transcription factors to prevent assembly of transcription initiation complex. -
Changing the chromatin structure
Bound repressor may recruit histone deacetylase and repressible chromatin remodeling complex to condense chromatin. This decreases the accessibility of general transcription factors and RNA polymerase to the promoter.
Describe the importance of 5’ cap to mature mRNA
(i) processing (mRNA splicing and polyadenylation)
-5’ cap helps the cell to recognise mRNA (amongst all other RNA molecules in the cell). Ensures subsequent steps such as splicing occurs on correct RNA molecule
(ii) Export out of nucleus
-5’ cap is recognised by certain proteins, which are required for the mRNA to exit from nucleus via nuclear pores.
(iii) half-life / stability
-5’ cap stabilises mRNA by protecting the growing pre-mRNA from rapid degradation by cellular ribonucleases.
(iv) translation
-5’ cap heps promote translation initiation. The cap is recognised by eukaryotic initiation factors. Binding of initiation factors to the cap helps recruitment of mRNA to small ribosomal subunit.
Describe how spliceosome results in alternative RNA splicing
- The introns are excised and exons are joined together/spliced together by spliceosomes; an snRNA (small nuclear RNA)-protein complex which recognise the sequences at intron-exon boundaries (points of excision) -> so that functional proteins can be produced
- Alternative splicing -> where different exons of a single pre-mRNA can be joined together such that different mature mRNAs and so ->different proteins can be produced
Describe how poly-A tail is added to 3’ end of mRNA and its importance.
-3’ end of pre-mRNA being cleaved enzymatically to make it shorter. Adenosine monophosphates are added one at a time to form a poly-A tail at 3’ end of mRNA.
-AMP added by enzyme poly-A-polymerase
Importance:
(i) enhances the half-life/stability of the mRNA transcript by slowing down its degradation by ribonucleases in nucleus and cytoplasm
(ii) serves as a signal to direct export of mature mRNA from nucleus to cytoplasm
(iii) interacts with initiation factors (together with 5’ cap) to form the translation initiation complex
Explain how the length of 3’ poly-A tail affects the half-life of mRNA
mRNA half-life is determined by the length of its poly-A tail. The longer the poly-A tail, the longer the mRNA can be used as a template to make proteins.
The poly-A tail is removed by ribonucleases in the 3’ to 5’ direction until a critical length is reached which will triggers removal of the 5’ cap and degradation of the mRNA from the 5’ end too.
Explain how anti-sense RNA works in silencing gene expression
-anti-sense RNA which is complementary to part of the mRNA to be degraded will be synthesised. It will complementary base pair with mRNA to form double stranded RNA
-This double-stranded RNA
1) is then targeted for degradation by ribonucleases
2) will block translation of the mRNA
Describe how a protein is targeted for degradation.
To tag a protein for degradatoin, an enzyme, ubiquitin ligase, will catalyse addition of a protein, ubiquitin, to the target protein.
-Ubiquitin-tagged protein is then recognised by a proteasome which can cleave this protein into smaller peptides that can be further degraded by enzymes in the cytoplasm.
