DNA and genes (L2-7) Flashcards
What is the structure of DNA?
2 helical anti-parallel polynucleotide chains made of 4 bases
What are the purines
Adenine and guanine
What are the pyrimidines
Thymine and cytosine
What are the nucleotide base pairings?
A + T (2 H-bonds formed)
G+C (3 H-bonds formed)
What is DNA replication?
semi-conservative
This means each of the newly synthesised double helix is made of 1 parent strand and 1 newly synthesised strand
Parental strand acts as a template for the synthesis of the new strand
Summarise DNA synthesis
It’s the sequential polymerisation of nucleotide building blocks by DNA Polymerase
Incoming nucleotides can only be added too the free hydroxyl on the 3’ carbon terminal Deoxyribose
What is the overall polarity of DNA strands
5’ to 3’, orientated antiparallel to each other
Formation of base pairs require 2 DNA strands to adopt an antiparallel orientation
Summary of the DNA replication mechanism
Synthesis of new strands initiated by formation of replication fork where strands are pulled apart/separated
At the replication fork, 2 new strands are synthesised and have antiparallel orientation as DNA synthesis is only in 5’->3’ direction
What is the role of DNA helicase
Used to pull apart DNA strands in the first steps of DNA synthesis
DNA Helicase continuously unwinds parent DNA to give DNA Primase and Polymerase access to DNA template sequence
What is the role of DNA polymerase
synthesising new strands
Why can the new strands NOT be synthesised continuously?
Due to the antiparallel orientation of the parent stands and unidirectional orientation of the new DNA strand
What is the leading strand
It is continuous and proceeds the lagging strand
What is the lagging strand?
It is discontinuous and made of Okazaki fragments
What is the role of RNA primer?
Initiate DNA synthesis by extension of RNA primer
All Okazaki fragments and leading strands are intitated as short RNA molecules by a special RNA polymerase DNA Primase
What is the role of single-stranded Binding Proteins (SSBs)
they maintain the unwound parent DNA strands in a single stranded confirmation to ease replication fork progression
When does Okazaki fragment synthesis stop?
When DNA Polymerase reaches RNA Primer @ the 5’ end of another fragment
What happens to the RNA Primers by the end?
They are extended by DNA Polymerase then removed by Ribonuclease H and the gap is filled by DNA Polymerase
What does DNA ligase do by the end of Replication?
It covalently links the Okazaki fragments of the lagging strand to a continuous DNA strand
What is gene expression?
Gene expression is the process by which the heritable information in a gene, the sequence of DNA base pairs, is made into a functional gene product, such as protein or RNA. The basic idea is that DNA is transcribed into RNA, which is then translated into proteins
What are the 2 main steps of gene expression
- Transcription
mRNA synthesis - Translation
protein synthesis
What is the structural features of RNA molecules
- RNA backbone is ribose sugars and not deoxyribose sugars
2. RNA transcripts contain Uracil instead of Thymine, Uracil pairs with Adenine and still forms 2 hydrogen bonds
How is transcription initiated in prokaryotic genes?
By binding of RNA polymerase to DNA sequences in the promoter region which lies upstream of the transcription start site
This defines the position at which synthesis of RNA transcripts begin
What is transcript elongation and what does it do?
The stage where the RNA strand gets longer
Done by RNA Polymerase which yields and mRNA transcript (this is a copy of the DNA sequence)
Transcripts are single stranded
They are synthesised in a 5’-3’ direction
How is transcription in prokaryotes terminated?
When RNA Polymerase encounters the terminator region which promotes dissociation of RNA Polymerase from the DNA
How is the transcription rate limited?
By the frequency of transcription inititation
Summarise transcription of eukaryotic genes
Similar to prokaryotic
Has 3 phases:
- Inititation
- Elongation
- Termination
RNA Splicing, what is it?
Primary transcripts of eukaryotic genes are made of segments, exons and introns
Introns are spliced out the primary RNA transcript and do not appear in mature mRNA
What are the major steps of gene expression regulation?
- Transcriptional control
- RNA Processing control
- RNA transport and localisation control
- Translation control
- mRNA degradation control
- protein activity control
How many RNA Polymerases carry out transcription of eukaryotic genes?
3:
RNA Polymerase I, II & III
RNA Polymerase II is important one
What does RNA Polymerase II do?
Its an essential workforce in the cell, it transcribed all protein coding RNAs.
It’s the most important polymerase
What are the types of protein coding genes?
Ubiquitously expressed (expressed everywhere and have a role in every cell) and deferentially expressed protein coding genes (differential genes)
What does levels of housekeeping gene expression (ubiquitously expressed) depend on
nutrient availability and rate of tissue growth and cell proliferation
What is the experimental technique of in situ hybridisation?
it detects where a gene is being transcribed in tissues and whole organisms
How is the transcriptional control triggered?
When the “trans-acting” transcription factor protein binds to a certain “cis-acting” DNA sequence next to the gene
What are cis-acting sequences?
sequences around the gene that are binding sites for proteins that regulate transcription
What are trans-acting factors?
proteins that bind to the cis acting sequence
Intitation of transcript synthesis requires physical attraction between what?
trans-acting factors
genral transcription factors
RNA polymerase
How is transcription inititated?
binding of GTFs to TATA box in the promoter DNA sequence
TATA binding proteins recognise the TATATA in a sequence and will form hydrogen bonds with these bases
GTFs are…
…ubiquitous and dont have a regulatory role
What do GTFs do during transcription
they help RNA Polymerase II bind to the transcription initiation site by distorting the DNA sequences of the promoter
This protein assembly on DNA is known as Transcription Inititation Complex (TIC)
TIC is…
inactive until it is contactd by Transcription Activator prtoein bound to a cis-acting sequence
TIC is formed at the 5’ end of gene
What are regulatory transcription factors?
a type of transcription factor
Bind to enhancer/cis-acting sequence
How is efficient trancriptional activation of a target gene achieved?
It requires other transcription activators and auxiliary proteins as well as TIC to decide WHERE the gene will be transcribed by:
stabilizing the TIC and
activating RNA Polymerase
*Activators stabalize TIC and promoter DNA sequence interaction long enough for RNA transcript to be synthesised)
3 examples of importance of transcription factors?
- Antennapedia transcription factor (Antp)
- promotes antennae development, mutation = legs not antennae - Cycloidea Transcription Factor
- Gli3 transcription factor
What are Antp?
sequence specific DNA binding proteins that regulate transcription via protein-protein interactions affecting TIC activity
What are CYC proteins? (Cycloidea)
sequence-specific DNA-binding protein which binds to DNA sequence in target gene promoters and regulate transcription
What is the Gli3 transcription factor
sequence-specific DNA binding protein that is a component of the Hedgehog signalling pathway and regulates transcription of Hedgehog pathway target genes
Transcription factors can be…
…activators or repressors
Competition in transcription
Activators and repressors compete for interaction with RNA Polymerase
Competition helps regulate transcription
What is the structure of transcription factors?
Modular structure, means distinct areas of the protein have distinct functions
As transcriptions factors are modular, they have more than one domain inc…
DNA binding domain
Activation/ repressor domain (these promote/prevent recruitment of TIC)
regulatory domain
What is the zinc finger domain?
- Type of transcription factor
- A protein molecule that can recognise 3 base-pairs of double stranded DNA
- Has zinc ion to stabalise structure
- More zinc fingers in protein = longer sequence they recognise
- Zinc ions interact w/ different amino acids
