Title: BIO311 Prokaryote Gene Expression Section 1 Overview of RNA Function
1BIO311 - Prokaryote Gene ExpressionSection
1Overview of RNA Function
- Jasper Rees
- Department of Biotechnology, UWC
- www.biotechnology.uwc.ac.za/teaching/BIO311
2Overview 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
3Central 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
4Replication
- 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
5Transcription
- 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
6Translation
- 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
7Flow of Genetic Information
- DNA stores information in genes
- Transcribed from template strand into mRNA
- Translated into protein from mRNA by ribosomes
8Central 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
9Genotype 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
10mRNA 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)
11mRNA 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
12RNA 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
13mRNA 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
14Prokaryote 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
15Eukaryote 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
16Eukaryote 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
175 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
18Poly 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
19mRNA 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
20Eukaryote organelle mRNA structure
- Single stranded
- Polycistronic (many ORFs)
- Unmodified 5 and 3 ends
- Transcribed and translated together
- Show similarity to prokaryote genes and
transcripts
21Transfer 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
22tRNA - general features
- Cloverleaf secondary structure with constant base
pairing - Trinucleotide anticodon
- Amino acid covalently attached to 3 end
23tRNA 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
24tRNA conserved structures
- Conserved bases, modified bases, secondary
structures (base pairing), CAA at 3 end - Variable bases, variable loop
25tRNA secondary structure
- Four basepaired arms
- Three single stranded loops
- Free 3 end
- Variable loop
- Conserved in all
- Living organisms
26tRNA 2D and 3D views
- Projection of cloverleaf structure, to ribbons
outline of 3D organisation of general tRNA
structure
27tRNA 3D ribbon - spacefill views
Spacefill View
Ribbon view
28tRNAs 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.
29Aminoacylation 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
30Information 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
31Ribosomes
- 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
32Ribosome 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
333D 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
3450S ribosomal subunit 3D structure
35Overview 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
36Ribosomes 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
37Polysomes - one mRNA, many ribosomes
38Polysomes in electron micrographs
39Transcription 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
40Overall translation cycle
41Translation and transcription are coupled in
prokaryotes
42Prokaryote 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
43Eukaryote 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