1. Prokaryote Gene Expression Section 1 Overview of RNA Function

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
Ribbon view
Spacefill 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, 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
Elongation

41. Translation and transcription are coupled in prokaryotes

42. Prokaryote mRNA life cycle
Life cycle is rapid
Synthesis is at about 40 bases per second
Synthesis of complete mRNA may take 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 >48 hours)
Translation is on polysomes
mRNA half life is 4 to > 24 hours in the cytoplasm