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Prokaryote Gene Expression Section 1 Overview of RNA Function.

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Presentation on theme: "Prokaryote Gene Expression Section 1 Overview of RNA Function."— Presentation transcript:

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 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 Mg 2+ 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


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