29. Gene Expression_an overview Flashcards
1
Q
what is the central dogma of DNA?
A
- replication of DNA required for inheritance of genetic information
– DNA polymerase catalyses DNA replication - cells use DNA as genetic material which encode proteins
– catalyse cellular processes - some viruses use RNA as genetic information
– upon infection of cells, replication of RNA occurs in infected cell - expression of genetic information mostly undirectional
– transcription of DNA yields RNA molecules
— can be used to generate protein sequences
– translation of RNA into protein always irreversible
2
Q
what is reverse transcription?
A
- genomes of retroviruses consist of single stranded RNA molecules
- during infective cycle
– RNA converted to single-stranded DNA
– by reverse transcriptase - single-stranded DNA converted to double stranded DNA
– incorperated into genome of infected cell - duplex DNA inherited like any other gene
– eg. HIV - use reverse transcriptase in molecular biology to convert RNA into DNA
– allows to use as template for polymerase chain reaction (PCR) - PCR uses DNA polymerase enzyme to exponentially amplify DNA
3
Q
what drives transcription?
A
- driven by RNA polymerase
– generation of RNA transcripts for protein synthesis - RNA transcript identical to one stran of DNA
– called coding strand (non-template)
– and complementary (template strand)
4
Q
what is the process of transcription?
A
- RNA polymerase binds to promoter
– upstream of start of gene - RNA polymerase moves in 5’ -> 3’ direction
– to synthesise RNA molecule - RNA polymerase will continue adding base pairs
– until reaches terminator sequence - upstream sequences
– sequences prior to start poin of gene expression - downstream sequences
– sequences after stop codon - either DNA strand could be used as template DNA
5
Q
what are transcription promoters?
A
- RNA polymerase
– will bind strongly to DNA only at promoter sequence - Sigma factor (sigma polypeptide)
– ensures RNA polymerase only initiates transcription at promoter site - Sigma factor
– confers RNA polymerase the ability to recognise specific binding sites - RNA polymerase will bind to different promoters at different frequencies
– frequency at which RNA pol. initiates transcription from specific promoter gives idea of strangth of promoter to drive transcription
6
Q
what are the different types of promoters?
A
- constitutive promoters
– always expressed - leaky promoters
– expressed to some degree at all times
– can be induced at high levels - inducing gene expression
– the additon of the inducer to initiate gene expression
7
Q
what is the promoter sequence?
A
- promoter sequences contain particular DNA sequences
– recognised by RNA polymerase - minimum sequence length that can function as promoter in bacteria
– 12 bp
– shorter sequence may occur by chance - minimum lengths of promoter sequences increases as genome sizes increase
- promoter sequence have conserved regions
8
Q
what are the transcription regulatory promoter sequences?
A
- conservative very short consensus sequences
– typical promoters for both eukaryotes and prokaryotes - bacterial promoter
– start point: -10 sequences; -35 sequence
9
Q
what are the eukaryotic regulatory sequences?
A
- GC box
- E box
- TATA box
- CAAT box
- OCT box
10
Q
how can one identify promoter sequences?
A
- sequence upstream of gene sequence of interest
– analysed computationally, ‘hunt’ for conserved sequence
– confirmed experimentally - cut DNA up into fragments
– use fragments to initiate gene expression of a reporter gene (green flourescent protein, yellow flourescent protein, luciferase)
11
Q
what is the translation of mRNA?
A
- principle classes of RNA involved in protein synthesis
– mRNA (messenger)
– tRNA (transfer)
– rRNA (ribosomal) - mRNA = translated into protein sequence
- tRNA and rRNA prodive components for protein synthesis
– tRNA = transfer of amino acids into peptide chain
– rRNA = forms part of catalytic site of ribosomes
– ribosomes = globular proteins
12
Q
what part do codons play in the translation of mRNA?
A
- methionine
– only amino acid with one codon - UAA, UAG, UGA
– stop codons
– ribosome pauses and falls off mRNA transcript - woble theory
– base pairing relaxed at third position
– some tRNA have inosine (I) in anticodon position; it can pair with U, C, A. Don’t need 61 tRNA molecules
13
Q
what is codon usage in the translation fo mRNA?
A
- organisms use different codons at different frequencies
– to encode amino acids in their proteins - codon optimisation
– changing the DNA sequence to enccode amino acid with preferred codon for species in which expression of gene is desires - eg. expression of human gene in E.coli
– change Arg codons to CGC / CGU
14
Q
how is mRNA translated?
A
- mRNA very unstable
– rapidly degraded - in eukaryotes modified at ends to increas stability
– 5’ cap: Guanine attached to transcription site (Adenine or G) by 5’-5’ triphosphate linakge
– 3’ poly(A) tail: 200 A residues added to poly(A) polymerase enzyme following transcription - mRNA cleaved to give mature products
15
Q
what is translation in prokaryotes?
A
- mRNA transcribed and translates in one cellular compartment
- ribosomes attach to mRNA before transcription completed
– eukaryotes, synthesis and maturation of mRNA occurs in nucleus and mature mRNA transported to cytosol for translation - mRNA survives for minutes
– eukaryotic survives hours - mRNA degraded from 5’ end
