Prokaryote Gene Expression Section 1 Overview of RNA Function
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Transcript of Prokaryote Gene Expression Section 1 Overview of RNA Function
Prokaryote Gene Expression
Section 1
Overview of RNA Function
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
“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
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
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
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
Flow of Genetic Information
DNA stores information in genes
Transcribed from template strand into mRNA
Translated into protein from mRNA by ribosomes
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
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
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)
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
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
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
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
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
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
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
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
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
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
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
tRNA - general features
Cloverleaf secondary structure with constant base pairing
Trinucleotide anticodon Amino acid covalently
attached to 3’ end
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
tRNA conserved structures
Conserved bases, modified bases, secondary structures (base pairing), CAA at 3’ end
Variable: bases, variable loop
tRNA secondary structure
Four basepaired arms
Three single stranded loops
Free 3’ end Variable loop Conserved in allLiving organisms
tRNA 2D and 3D views
Projection of cloverleaf structure, to ribbons outline of 3D organisation of general tRNA structure
tRNA 3D ribbon - spacefill views
Ribbon view Spacefill View
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.
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
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
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
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
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
50S ribosomal subunit 3D structure
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
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
Polysomes - one mRNA, many ribosomes
Polysomes in electron micrographs
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
Overall translation cycle
Elongation
Translation and transcription are coupled in prokaryotes
Prokaryote mRNA life cycle
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
Eukaryote mRNA lifecycle
Transcription, capping, polyA, splicing are nuclear
Translation is cytoplasmic mRNA is complete before
export to cytoplasm (20 min to >48 hours)
Translation is on polysomes mRNA half life is 4 to > 24
hours in the cytoplasm