Download Prokaryote Gene Expression Section 1 Overview of RNA

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 anticodon 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 all
Living 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 exoRNAase 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