Prokaryote Gene Expression Section 1 Overview of RNA Function

1. Prokaryote Gene ExpressionSection 1Overview 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 Spacefill View Ribbon 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, 24-60 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. Elongation Overall translation cycle

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 1 - 5 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