1 CA Garca Seplveda MD PhD mRNA, tRNA y rRNA Laboratorio de Genmica Viral y Humana Facultad de Medicina, Universidad Autnoma de San Luis Potos 2 Contents Central Dogma mRNA Prokaryote Eukaryote Extrachromosomal (Mitochondrial) tRNA Translation mRNA Life Cycle Polycistronic and monocistronic mRNAs Prokaryotic and eukaryotic mRNAs 3 Central Dogma Genetic information is expressed in two stages: 1.- Transcription 2.- Translation Generation of ssRNA from DNA template Catalysed by RNA Polymerases Generates: mRNA tRNA rRNA Occurs in prokaryotes and eukaryotes by essentially identical processes 4 Central Dogma Genetic information is expressed in two stages: Synthesis of a protein mRNA as a template tRNAs convert codon information into amino acid sequence Catalysed by rRNA 1.- Transcription 2.- Translation 5 mRNA 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 6 mRNA 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 7 mRNA 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 8 mRNA 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 9 mRNA 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 10 mRNA 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 11 mRNA Prokaryotes They can code for one or many proteins (translation of products) in prokaryotes (polycistronic) 12 mRNA Eukaryotes They encode only one peptide(each) in eukaryotes (monocistronic). Polyproteins are observed in eukaryotic viruses, but these are a single translation product, cleaved into separate proteins after translation. 13 mRNA Synthesis (transcription) Catalysed by RNA Polymerase Stages: 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 14 mRNA Synthesis (Prokaryotes) 5 and 3 ends are unmodified Ribosomes bind at ribosome binding site (do not require a free 5 end) Can contain many open reading frames (ORFs) Translated 5 3 Transcribed and translated together 15 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 Production Degradation 16 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 Production Degradation 17 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 Production Degradation 18 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 Production Degradation 19 mRNA, Eukaryotes 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 20 Translation in Eukaryotes Translated from 5 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 21 Translation in Eukaryotes Translated from 5 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 22 Translation in Eukaryotes Translated from 5 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 23 mRNA, 5' Capping } 30 nm The 5' cap is a specially altered nucleotide end to the 5' end of precursor messenger RNA and, as a special exception, caliciviruses (Norwalk virus). Actue epidemic gastroenteritis (nursery, hospital and cruises). 24 mRNA, 5' Capping Consists of a guanine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage. This guanosine is methylated on C7 position directly after capping by a methyl transferase. It is referred to as a 7-methylguanosine cap,abbreviated m7G. 25 mRNA, 5' Capping Further modifications include the methylation of the 2' hydroxy-groups of the first 3 ribose sugars of the 5' end of the mRNA. Functionally the 5' cap looks like the 3' end of an RNA molecule (the 5' carbon of the cap ribose is bonded, and the 3' unbonded). Offers resistance to 5' exonucleases. 26 mRNA, 5' Capping process 1)One of the terminal phosphate groups is removed (phosphatase), leaving two terminal phosphates. 2)GTP is added to the terminal phosphates (guanylyl transferase), losing two phosphate groups (from the GTP) in the process. This results in the 5' to 5' triphosphate linkage. 3)The guanine is methylated (by a methyl transferase). 4)Other methyltransferases are optionally used to carry out methylation of 5' proximal nucleotides. 27 mRNA, 5' Capping process 1)One of the terminal phosphate groups is removed (phosphatase), leaving two terminal phosphates. 2)GTP is added to the terminal phosphates (guanylyl transferase), losing two phosphate groups (from the GTP) in the process. This results in the 5' to 5' triphosphate linkage. 3)The guanine is methylated (by a methyl transferase). 4)Other methyltransferases are optionally used to carry out methylation of 5' proximal nucleotides. 28 mRNA, 5' Capping process 1)One of the terminal phosphate groups is removed (phosphatase), leaving two terminal phosphates. 2)GTP is added to the terminal phosphates (guanylyl transferase), losing two phosphate groups (from the GTP) in the process. This results in the 5' to 5' triphosphate linkage. 3)The guanine is methylated (by a methyl transferase). 4)Other methyltransferases are optionally used to carry out methylation of 5' proximal nucleotides. 29 mRNA, 5' Capping process 1)One of the terminal phosphate groups is removed (phosphatase), leaving two terminal phosphates. 2)GTP is added to the terminal phosphates (guanylyl transferase), losing two phosphate groups (from the GTP) in the process. This results in the 5' to 5' triphosphate linkage. 3)The guanine is methylated (by a methyl transferase). 4)Other methyltransferases are optionally used to carry out methylation of 5' proximal nucleotides. 30 mRNA, 5' Capping process 1)The Capping Enzyme Complex (CEC) is bound to the RNA polymerase II before transcription starts. 2)As soon as the 5' end of the new transcript emerges the enzymes transfer to it and caps it. 3)The enzymes for capping can only bind to RNA polymerase II. 31 mRNA, 3' Polyadenylation Covalent linkage of a poly(Adenine) tail to most mRNA (eukaryotic). The poly-A tail protects the mRNA from exonucleases and is important for transcription termination, for export of the mRNA from the nucleus, and for translation. Some prokaryotic mRNAs also are polyadenylated. Polyadenylation occurs after transcription of DNA into RNA in the nucleus. Poly-A signal is transcribed, mRNA is cleaved (endonuclease) in a special site (AAUAAA) then 50 to 250 adenine residues are added. This reaction is catalyzed by polyadenylate polymerase. 32 mRNA, 3' Polyadenylation Cleavage and Polyadenylation Specificity Factor (CPSF) & Cleavage Stimulation Factor (CstF), both of which are multi- protein complexes, start bound to the rear of the advancing RNA polymerase II. 33 mRNA, 3' Polyadenylation As RNA polymerase II advances over the adenylation signal sequence, CPSF & CstF transfer to the pre-mRNA. CPSF binds to the AAUAAA sequence. CstF binds to the GU or U rich sequence following it. 34 mRNA, 3' Polyadenylation CPSF & CstF promote cleavage 35 nt after AAUAAA sequence. Polyadenylate Polymerase (PAP) immediately starts adding Adenines. Cleavege does not take place before PAP has bound. Nuclear Polyadenylate Binding Protein (PABPN1) immediately binds to the new poly-A tag. 35 mRNA, 3' Polyadenylation PAB displaces CPSF. PAP continues to add 100 250 adenines (depending on the organism). PABPN1 acts as a molecular ruler, specifying when polyadenylation should stop. 36 Mitochondrial mRNA = Prokaryote Single stranded Polycistronic (many ORFs) Unmodified 5 and 3 ends Transcribed and translated together Show similarity to prokaryote genes and transcripts 37 tRNA tRNA first hypothesized by Francis Crick. Small RNA chain that transfers a specific amino acid to a growing polypeptide chain in the ribosome Has a 3' terminal site for amino acids (whose linkage depends on aminoacyl tRNA synthetase). Contains a three base region called the anticodon that complements the codon on the mRNA. Each type of tRNA molecule can be attached to only one type of amino acid. tRNA molecules bearing different anticodons may also carry the same amino acid (degenerecy). 38 tRNA Structure tRNA has primary structure (sequence), secondary structure (cloverleaf), and tertiary structure (L-shape). 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 39 tRNA Structure 1)The 5'-terminal phosphate group. 2)The acceptor stem (7bp) composed by the 5'-terminus base paired to the 3'-terminus (contains non-Watson- Crick base pairs). 3)The CCA tail is at the 3' end of the tRNA molecule (important for the recognition of tRNA by enzymes critical in translation). NOTE: In prokaryotes, the CCA sequence is transcribed. In eukaryotes, the CCA sequence is added. 4)The D arm (18bp) ends in a loop which contains dihydrouridine. 5)The anticodon arm (ca17bp) contains the anticodon. 6)The T arm (17bp) contains T C sequence ( = pseudouridine). 7)Modified (methylated) bases occur in several positions outside the anticodon. First anticodon base sometimes modified to inosine or ). 40 tRNA Structure 41 tRNA Codons & Anti-codons An anticodon is a unit made up of three nucleotides that complement the three bases of the codon on the mRNA. 42 tRNA Codons & Anti-codons Different base triplets (codons) in mRNA code for different amino acids. 43 tRNA Codons & Anti-codons The Genetic Code is degenerate 44 tRNA Codons & Anti-codons The Genetic Code is degenerate Serine can be: UCU, UCC, UCA, UCG, AGU & AGC Each tRNA contains a specific anticodon triplet sequence that can base-pair to one or more codons for an amino acid. For example, one codon for Serine is AGU; the anticodon being UCA. Some anticodons can pair with more than one codon due to wobble base pairing. When the first nucleotide of the anticodon is either inosine or pseudouridine (can hydrogen bond to different bases). 45 tRNA Aminoacylation Aminoacylation is the process of covalently adding an aminoacyl group to a compound tRNA). Each tRNA is aminoacylated (charged) with a specific amino acid by an aminoacyl tRNA synthetase. There is normally a single aminoacyl tRNA synthetase for each amino acid. There can be more than one tRNA, and more than one anticodon, for an amino acid. Recognition of the tRNA by the synthetases depends on the anticodon, and the acceptor stem. 46 tRNA genes Organisms have varying amounts of tRNA genes. C. elegans has 29,647 genes of which 620 code for tRNA. Saccharomyces cerevisiae has 275 tRNA genes in its genome. In the human genome there are: 4,421 non-coding RNA genes (which include tRNA genes). 22 mitochondrial tRNA genes 497 nuclear genes encoding cytoplasmic tRNA molecules and 324 tRNA-derived putative pseudogenes. 47 tRNA genes Cytoplasmic tRNA genes are grouped into 49 families according to their anti- codon features. tRNA genes are found on all chromosomes, except 22 and Y. High clustering on 6p and 1 is observed (140 tRNA genes). tRNA molecules are transcribed (in eukaryotic cells) by RNA polymerase III, unlike messenger RNA which is transcribed by RNA polymerase II. pre-tRNAs contain introns; in bacteria these self-splice, whereas in eukaryotes and archaea they are removed by tRNA splicing endonuclease. 48 Ribosomes Prokaryotic ribosomes are smaller than most of the eukaryotes. The ribosomes in eukaryote mitochondria resemble those in bacteria. The function of ribosomes is the assembly of proteins (translation). Ribosomes catalyze the assembly of individual amino acids into polypeptide chains. They use mRNA as a template to join a correct sequence of amino acids. This reaction uses adapters called tRNA. 49 Ribosomes First observed in the mid-1950s by Romanian cell biologist George Palade using an electron microscope as dense particles or granules Nobel Physiology, The term "ribosome" was proposed by scientist Richard B. Roberts in 1958 Ribonucleic body (soma) 50 Ribosomes All ribosomes are composed of two subunits that separate when translation terminates and reunite when an new initiation complex is formed. Small subunit Large subunit mRNA tRNA 51 Session #20-22 Translation Introduction The basic form of the ribosome is conserved, but there are appreciable variations in the overall size and proportions of RNA and protein in the ribosomes of bacteria, eukaryotic cytoplasm, and organelles. Al ribosomes consists of two subunits, each of which contains a major rRNA and a number of small proteins. The large subunit may also contain smaller RNAs. 52 Svedberg Units Briefly Theodor Svedberg ( ). Nobel laureate (Chemistry,1926) for his work on colloids and ultracentrifugation. Colloid = Mechanical mix (milk) with a dispersed and continuous phase. Svedberg, non-SI unit used to characterize the behaviour of a particle in ultracentrifugation. S = unit of time amounting to s or 100 femtoseconds. S not additive, since the sedimentation rate is associated with the shape & size of the particle. When two particles bind together there is a loss of surface area, when measured separately they will have Svedberg values that do not add up. 53 Session #20-22 Translation Introduction The ribosomal proteins are known as r-proteins. With the exception of one protein present at four copies per ribosome, there is one copy of each protein. 54 Session #20-22 Translation Phylogenetics The ribosomes of higher eukaryotes are larger than those of bacteria. Total content of both RNA & protein is greater Major RNA molecules are longer (18S & 28S rRNAs) Possess more proteins. RNA is the predominant component. 55 Session #20-22 Translation Phylogenetics The ribosomes of higher eukaryotic cytoplasm are larger than those of bacteria. Total content of both RNA & protein is greater Major RNA molecules are longer (18S & 28S rRNAs) Possess more proteins. RNA is the predominant component. 56 Session #20-22 Translation Phylogenetics The ribosomes of higher eukaryotic cytoplasm are larger than those of bacteria. Total content of both RNA & protein is greater Major RNA molecules are longer (18S & 28S rRNAs) Possess more proteins. RNA is the predominant component. 57 Session #20-22 Translation Phylogenetics In prokaryotes: Ribosomes = 2.5 MDa rRNA = 66% of the ribosomal mass In eukaryotes (mammals): Ribosomes = 4.2 MDa rRNA = 60% of the ribosomal mass Organelle ribosomes are distinct from eukaryotic cytosol ribosomes and take varied forms. In some cases, they are almost the size of bacterial ribosomes and have 70% RNA; in other cases, they are only 60S and have