BIO311 Prokaryote Gene Expression Section 1 Overview of RNA Function

1
BIO311 - Prokaryote Gene ExpressionSection
1Overview of RNA Function

• Jasper Rees
• Department of Biotechnology, UWC
• www.biotechnology.uwc.ac.za/teaching/BIO311

2
Overview Section 1

• Central Dogma of molecular biology
• mRNA Structure and organisation
• Prokaryotic mRNA
• Eukaryotic cytoplasmic mRNA
• Eukaryotic organelle mRNA
• tRNA structure and overview of function
• Overview of translation
• Biosynthetic cycle of mRNA
• Polycistronic and monocistronic mRNAs
• Prokaryotic and eukaryotic mRNAs

3
Central Dogma of molecular biology

• dogma - a strongly held viewpoint or idea
• Genetic information is stored in DNA, but is
  expressed as proteins, through the intermediate
  step of mRNA
• The processes of Replication, Transcription and
  Translation regulate this storage and expression
  of information

4
Replication

• Process by which DNA (or RNA) is duplicated from
  one molecule into two identical molecules
• Semi conservative process resulting in two
  identical copies each containing one parental and
  one new strand of DNA
• Catalysed by DNA polymerases
• Process essentially identical between prokaryotes
  and eukaryotes

5
Transcription

• Generation of single stranded RNA from a DNA
  template (gene)
• Catalysed by RNA Polymerases
• Generates
• mRNA - messenger RNA
• tRNA - transfer RNA
• rRNA - ribosomal RNA
• Occurs in prokaryotes and eukaryotes by
  essentially identical processes

6
Translation

• The synthesis of a protein sequence
• Using mRNA as a template
• Using tRNAs to convert codon information into
  amino acid sequence
• Catalysed by ribosomes
• Process essentially identical between prokaryotes
  and eukaryotes

7
Flow of Genetic Information

• DNA stores information in genes
• Transcribed from template strand into mRNA
• Translated into protein from mRNA by ribosomes

8
Central Dogma

• Information in nucleic acids (DNA or RNA) can be
  replicated or transcribed. Information flow is
  reversible
• However, there is no flow of information from
  protein back to RNA or DNA

9
Genotype and Phenotype

• A Genotype is the specific allele at a locus
  (gene). Variation in alleles is the cause of
  variation in individuals
• mRNA is the mechanism by which information
  encoded in genes is converted to proteins
• The activities of proteins are responsible for
  the phenotype attributable to a gene
• The regulation of the level of expression of mRNA
  is therefore the basis for regulating the
  expression of the phenotype of a gene
• Regulation is primarily at the level of varying
  the rate of transcription of genes

10
mRNA Structure

• mRNAs are single stranded RNA molecules
• They are copied from the TEMPLATE strand of the
  gene, to give the SENSE strand in RNA
• They are transcribed from the 5 to the 3 end
• They are translated from the 5 to the 3 end
• Generally mRNAs are linear (although some
  prokaryotic RNA viruses are circular and act as
  mRNAs)

11
mRNA information coding

• They can code for one or many proteins
  (translation of products) in prokaryotes
  (polycistronic)
• They encode only one protein (each) in eukaryotes
  (monocistronic)
• Polyproteins are observed in eukaryotic viruses,
  but these are a single translation product,
  cleaved into separate proteins after translation

12
RNA synthesis

• Catalysed by RNA Polymerase
• Cycle requires initiation, elongation and
  termination
• Initiation is at the Promoter sequence
• Regulation of gene expression is at the
  initiation stage
• Transcription factors binding to the promoter
  regulate the rate of initiation of RNA Polymerase

13
mRNA life cycle

• mRNA is synthesised by RNA Polymerase
• Translated (once or many times)
• Degraded by RNAses
• Steady state level depends on the rates of both
  synthesis and degradation

14
Prokaryote mRNA structure

• Linear RNA structure
• 5 and 3 ends are unmodified
• Ribosomes bind at ribosome binding site,
  internally within mRNA (do not require a free 5
  end)
• Can contain many open reading frames (ORFs)
• Translated from 5 end to 3 end
• Transcribed and translated together

15
Eukaryote cytoplasmic mRNA structure

• Linear RNA structure
• 5 and 3 ends are modified
• 5 GpppG cap
• 3 poly A tail
• Transcribed, spliced, capped, poly Adenylated in
  the nucleus, exported to the cytoplasm

16
Eukaryote mRNA translation

• Translated from 5 end to 3 end in cytoplasm
• Ribosomes bind at 5 cap, and do require a free
  5 end
• Can contain only one translated open reading
  frames (ORF). Only first open reading frame is
  translated

17
5 cap structures on Eukaryote mRNA

• Caps added enzymatically in the nucleus
• Block degradation from 5 end
• Required for RNA spicing, nuclear export
• Binding site for ribosomes at the start of
  translation

18
Poly A tails on eukaryote mRNA

• Added to the 3 end by poly A polymerase
• Added in the nucleus
• Approximately 200 A residues added in a template
  independent fashion
• Required for splicing and nuclear export
• Bind poly A binding protein in the cytoplasm
• Prevent degradation of mRNA
• Loss of poly A binding protein results in sudden
  degradation of mRNA in cytoplasm
• Regulates biological half-life of mRNA in vivo

19
mRNA Splicing

• Eukaryote genes made up of Exons and Introns
• mRNA transcripts contain both exons and introns
  when first synthesised
• Intron sequences removed from mRNA by Splicing in
  the nucleus
• Occurs in eukaryotes, but not in prokaryotes
• Alternative splicing can generate diversity of
  mRNA structures from a single gene

20
Eukaryote organelle mRNA structure

• Single stranded
• Polycistronic (many ORFs)
• Unmodified 5 and 3 ends
• Transcribed and translated together
• Show similarity to prokaryote genes and
  transcripts

21
Transfer RNA

• Small RNAs 75 - 85 bases in length
• Highly conserved secondary and tertiary
  structures
• Each class of tRNA charged with a single amino
  acid
• Each tRNA has a specific trinucleotide anti-codon
  for mRNA recognition
• Conservation of structure and function in
  prokaryotes and eukaryotes

22
tRNA - general features

• Cloverleaf secondary structure with constant base
  pairing
• Trinucleotide anticodon
• Amino acid covalently attached to 3 end

23
tRNA constant bases and base pairing

• Constant structures of tRNAs due to conserved
  bases at certain positions
• These form conserved base paired structures which
  drive the formation of a stable fold
• First four double helical structures are formed
• Then the arms of the tRNA fold over to fold the
  3D structure
• The formation of triple base pairings stabilise
  the overall 3D structure

24
tRNA conserved structures

• Conserved bases, modified bases, secondary
  structures (base pairing), CAA at 3 end
• Variable bases, variable loop

25
tRNA secondary structure

• Four basepaired arms
• Three single stranded loops
• Free 3 end
• Variable loop
• Conserved in all
• Living organisms

26
tRNA 2D and 3D views

• Projection of cloverleaf structure, to ribbons
  outline of 3D organisation of general tRNA
  structure

27
tRNA 3D ribbon - spacefill views
Spacefill View
Ribbon view
28
tRNAs have common 3D structure

• All tRNAs have a common 3D fold
• Bind to three sites on ribosomes, which fit this
  common 3D structure
• Function to bind codons on mRNA bound to ribosome
  and bring amino acyl groups to the catalytic site
  on the ribosome
• Ribosomes to not differentiate tRNA structure or
  amino acylation.

29
Aminoacylation of tRNAs

• tRNAs have amino acids added to them by enzymes
• These enzymes are the aminoacyl tRNA synthetases
• They add the specific amino acid to the correct
  tRNA in an ATP dependent charging reaction
• Each enzyme recognises a specific amino acid and
  its cognate tRNA, but does not only use the
  anti-codon for the specificity of this reaction
• There are 20 amino acids, 24-60 tRNAs and
  generally approximately than 20 aa-tRNA
  synthetases

30
Information content and tRNAs

• The information in the mRNA in decoded by the
  codon-anti-codon interaction in ribosome
• The amino acid is not important, as the
  specificity of addition of the amino acid is at
  the charging step by the aa tRNA synthetase

31
Ribosomes

• Highly conserved structures
• Found in all living organisms
• Made of RNA and ribosomal proteins
• Have two subunits, which bind together to protein
  synthesis
• Cycle of protein synthesis consists of
  Initiation, Elongation and Termination

32
Ribosome structure

• Two subunits
• 50S and 30S in prokaryotes
• 60S and 40S in eukaryotes
• In dynamic equilibrium
• Association in Mg2 dependent in vitro
• In vivo cycle depends on protein factors

33
3D structure of ribosomes

• Most complex macromolecular complex yet
  characterised
• Atomic resolution structure provides much
  information about mechanisms of binding
  substrates, and mechanisms of catalysis
• Is helping to clarify mechanisms of action of
  antibiotics, which will lead to improved drug
  designs in future

34
50S ribosomal subunit 3D structure
35
Overview of Translation

• Biosynthesis of polypeptide (protein)
• Requires information content from mRNA
• Catalysed by ribosomes
• Requires amino acyl-tRNAs, mRNA, various protein
  factors, ATP and GTP
• Rate of translation of mRNA determined by rate of
  initiation of translation of mRNA
• Translation is not generally used as a regulatory
  point in control of gene expression

36
Ribosomes recycle in protein synthesis

• Ribosomes available in a free pool in cytoplasm
• Bind to mRNA at initiation of translation
• After termination are released from mRNA and
  recycled for further translation

37
Polysomes - one mRNA, many ribosomes
38
Polysomes in electron micrographs
39
Transcription and translation

• RNA and protein synthesis are coupled processes
  in prokaryotes
• As soon as the 5 end of the mRNA is
  biosynthesised it is available for translation
• Ribosomes bind, and start protein synthesis
• Degradation of the mRNA starts from the 5 end
  through exo-RNAase action
• The 5 end can be degraded before the 3 end is
  synthesised
• Coupling of these processes is important for
  regulation of gene expression

40
Overall translation cycle
41
Translation and transcription are coupled in
prokaryotes
42
Prokaryote mRNA lifecycle

• Life cycle is rapid
• Synthesis is at about 40 bases per second
• Synthesis of complete mRNA may take 1 - 5 minutes
• Translation and degradation occur with similar
  rates

43
Eukaryote mRNA lifecycle

• Transcription, capping, polyA, splicing are
  nuclear
• Translation is cytoplasmic
• mRNA is complete before export to cytoplasm (20
  min to gt48 hours)
• Translation is on polysomes
• mRNA half life is 4 to gt 24 hours in the cytoplasm