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