29. Gene Expression_an overview

Question 1

what is the central dogma of DNA?

Answer 1

• 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

Question 2

what is reverse transcription?

Answer 2

• 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

Question 3

what drives transcription?

Answer 3

• 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)

Question 4

what is the process of transcription?

Answer 4

• 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

Question 5

what are transcription promoters?

Answer 5

• 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

Question 6

what are the different types of promoters?

Answer 6

• 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

Question 7

what is the promoter sequence?

Answer 7

• 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

Question 8

what are the transcription regulatory promoter sequences?

Answer 8

• conservative very short consensus sequences
  – typical promoters for both eukaryotes and prokaryotes
• bacterial promoter
  – start point: -10 sequences; -35 sequence

Question 9

what are the eukaryotic regulatory sequences?

Answer 9

• GC box
• E box
• TATA box
• CAAT box
• OCT box

Question 10

how can one identify promoter sequences?

Answer 10

• 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)

Question 11

what is the translation of mRNA?

Answer 11

• 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

Question 12

what part do codons play in the translation of mRNA?

Answer 12

• 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

Question 13

what is codon usage in the translation fo mRNA?

Answer 13

• 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

Question 14

how is mRNA translated?

Answer 14

• 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

Question 15

what is translation in prokaryotes?

Answer 15

• 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

Question 16

what are eukaryotic genes?

Answer 16

• eukaryotic genes contain introns
  – waste of DNA
• prokyotes lost intron through evolution
  – eukaryotes not yet
• may be site for chromosome recombination to occur
  – does not alter integrity of coding sequence of gene
• alternative splicing of pre-mRNA
  – may yield slight variation to gene products
  – help regulate development of multicellular organisms

Question 17

how is the lac operon expressed in prokaryotic genes?

Answer 17

• genome of Escherichia coli
  – contain 4000 genes coding for functional proteins
  – not all expressed at same time
• E.coli gene expression regulated based on food sources available
• lac operon
  – group of genes functionally related to lactose metabolism

Question 18

what is the lac operon in E.coli?

Answer 18

• E.coli carbon source
  – monosaccharide glucose
  – disaccharide lactose
• lactose hydrolysed/digested into monomeric units before use
• absence of glucose
  – lac operon encoding genes for lactose processing are induced
  – E.coli rather use glucose if available

Question 19

what is the expression system used in molecular biology?

Answer 19

• IPTG
  – isopropyl Beta-D-1-thiogalactopyranoside
  – inducer of lac operon
  – used in media to enhance LacZ expression

Question 20

why choose IPTG than allolactose?

Answer 20

• IPTG not part of metabolic pathways
  – not broken down/used by cell
• ensures concentration of IPTG remains constant
  – thus, more useful inducer of lac operan than lactose itself

Question 21

what do we need recombinant gene/transgene expression for?

Answer 21

• DNA template
• promoter
• transcription machinery
• translational machinery
• re-location of protein/catalyst to desired site