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Chapter 17 From Gene to Protein Overview: The Flow of Genetic Information • the information content of DNA is in the form of specific sequences of nucleotides • the DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins • proteins are the links between genotype and phenotype • Gene expression = process by which DNA directs protein synthesis – includes two stages: transcription and translation Basic Principles of Transcription and Translation • RNA is the bridge between genes and the proteins for which they code • Transcription = synthesis of RNA – using information in DNA • Translation = synthesis of a polypeptide – using information in the mRNA – Ribosomes - sites of translation DNA RNA Protein The Products of Gene Expression: A Developing Story • original hypothesis posed by scientists: one gene – one enzyme • BUT a lot of proteins aren’t enzymes - researchers later revised the hypothesis: one gene–one protein • many proteins are composed of several polypeptides – each of which has its own gene • can now restated the hypothesis as the one gene–one polypeptide hypothesis • **Note: common to refer to gene products as proteins rather than polypeptides DNA vs. RNA Types of RNA mRNA = messenger RNA – majority of RNA found in a cell – carries the genetic information which will be translated into a protein sequence – defined by the presence of a “cap” at its 5’ end and a long tail of adenines at its 3’ end = “poly-A tail” Types of RNA rRNA = ribosomal RNA found in the nucleolus combines together with the large and small ribosomal subunits to form the functional ribosome (protein translation) rRNA is transcribed in the nucleolus by RNA polymerase I 28S rRNA Types of RNA tRNA = transfer RNA actually translates the message coded in the mRNA into a protein sequence which will become a function protein tRNA is transcribed in the nucleoplasm by an enzyme called RNA polymerase III then exported into the cytoplasm where AA are added 5’ 3’ 3’ 5’ -transcription of RNA is similar to DNA replication – RNA is made in the 5’ to 3’ direction -enzyme called an RNA polymerase binds to only one of the DNA strands = the anti-sense (template strand) -it moves along the template DNA strand (in the 3’ to 5’ direction) and reads the nucleotide and adds a complementary RNA base - a growing strand of RNA complementary to the DNA strand results -BUT rather than a T being paired with an A – U becomes the partner to A Transcription -a human gene is also known as a transcription unit = stretch of DNA that is transcribed into RNA -a transcription units is comprised of: 1. coding sequence – gives rise to protein strand upon translation -contains regions of code = “exons” – code for amino acids -and regions of junk = “introns” – spliced out in the nucleus 5’ 3’ Exon Intron Exon Intron Exon Intron Exon Transcription - 2. untranslated regions (UTRs) - the regions upstream and downstream of the coding region that are transcribed but NOT translated into a protein - -play an important role in translation – can influence the binding of the ribosome to the mRNA - -also play a role in exporting the mRNA into the cytoplasm Transcription • genes are also associated with additional sequences of DNA 1. core promoter sequence – for the binding of the RNA polymerase -RNA polymerase recognizes specific sequences of nt’s -binding is helped out by transcription factors 2. enhancer regions – help enhance transcription can be several thousands of base pairs upstream of the gene Transcription • • • • the transcription unit is transcribed by an RNA polymerase three types of RNA polymerase – I, II and III RNA polymerases create an RNA strand called a primary transcript • must be modified to produce the final mRNA, tRNA or rRNA RNA polymerase II transcribes protein coding genes into a primary transcript called premRNA – this is then is processed into mRNA – genes for tRNA are transcribed in the cytoplasm by RNA polymerase III – primary transcript is modified into tRNA – genes for rRNA is transcribed in the nucleolus by RNA polymerase I – primary transcript is modified into rRNA -3D representation of the RNA polymerase II enzyme Transcription • three stages of transcription – Initiation: binding of the RNA polymerase to the promoter • special sequences denote this region – Elongation: movement of the RNA polymerase along the anti-sense DNA strand and synthesis of the RNA transcript – Termination: release of the RNA polymerase from the DNA • special sequences denote this region • differs between prokaryotes and eukaryotes Promoter Transcription unit 5 3 Start point RNA polymerase DNA 3 5 1. Initiation – RNA polymerase binds to a special sequence of nucleotides called the promoter -certain sections of the promoter are important in 1 A eukaryotic promoter polymerase binding = core promoter Promoter Nontemplate strand DNA -in prokaryotes the promoter binds the RNA 5 3 5 3 polymerase without help TATA box Template strand Start point 2 Several transcription -in eukaryotes – the polymerase requires the assistance Transcription factors bind to DNA factors of proteins called transcription factors -specific transcription factors bind to the promoter 5 3 3 5 first and then help position the polymerase at the 3 Transcription initiation promoter complex forms -additional transcription factors then bind RNA polymerase II Transcription factors -entire complex is called the Transcription Initiation 5 3 Complex 3 5 3 TAT AAAA AT AT T T T 5 RNA transcript Transcription initiation complex sequence given in texts is that of the sense strand Promoter Transcription unit 5 3 Start point RNA polymerase DNA 3 5 1 Initiation Nontemplate strand of DNA 3 5 5 3 Unwound DNA RNA transcript Template strand of DNA 1. Initiation cont… -RNA polymerase unwinds the DNA helix (acts as a helicase) – exposes about 10 to 20 nucleotides for copying -RNA polymerase holds the DNA helix open (acts like the SSBs) -RNA polymerase initiates RNA synthesis without the need for a primer Promoter Transcription unit 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation Nontemplate strand of DNA 3 5 5 3 Unwound DNA RNA transcript 2. Elongation – RNA polymerase synthesizes a complementary RNA strand -RNA primary transcript grows in the 5’ to 3’ direction -uses uracil instead of thymine -the DNA strands reform their helix once the RNA polymerase moves past the area -the mRNA strand emerges from the polymerase-DNA complex Template strand of DNA 2 Elongation Rewound DNA 5 3 3 5 Nontemplate strand of DNA RNA nucleotides 3 5 RNA transcript RNA polymerase A T C C A A 3 C 5 3 end C A U C C A 5 Multiple RNA polymerases per DNA template 5 T A G G T T Direction of transcription Template strand of DNA Newly made RNA 3 Promoter Transcription unit 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation Nontemplate strand of DNA 3 5 5 3 Unwound DNA RNA transcript Template strand of DNA 2 Elongation Rewound DNA 5 3 3 5 3 5 RNA transcript 3 Termination 3 5 5 3 5 Completed RNA transcript 3 Direction of transcription (“downstream”) 3. Termination – RNA polymerase reaches a specific sequence of nucleotides and stops transcription -the RNA polymerase detaches from the DNA -the pre-RNA primary transcript is released -in prokaryotes – a termination sequence that detaches the polymerase -in eukaryotes – the RNA polymerase transcribes a sequence called a polyadenylation signal – for the release of the pre-RNA from the polymerase Transcription • to modify the primary transcript into mRNA – the following modifications are made: – a 5’methylated cap is added to the 5’end – addition of a 3’ poly A tail – the coding sequence is “edited” = splicing Eukaryotic cells modify RNA after transcription • enzymes in the eukaryotic nucleus modify pre-mRNA before exporting the mRNA to the cytoplasm – known as RNA processing • 5’ methylated cap – plays a role in the docking of the ribosome to mRNA – for translation – modified guanine nucleotide added after the transcription of about 20 to 40 nucleotides 5 G Protein-coding segment P P P 5 Cap 5 UTR Polyadenylation signal AAUAAA Start codon Stop codon 3 UTR 3 AAA … AAA Poly-A tail Eukaryotic cells modify RNA after transcription • 3’ poly A tail – plays a role in the export of the mRNA into the cytoplasm – after transcription – an enzyme adds 20 to 250 adenine nucleotides after the poly-adenylation signal sequence – also prevents degradation of the mRNA once its in the cytoplasm 5 G Protein-coding segment P P P 5 Cap 5 UTR Polyadenylation signal AAUAAA Start codon Stop codon 3 UTR 3 AAA … AAA Poly-A tail RNA Splicing • most eukaryotic genes and pre-RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions – the noncoding regions are called intervening sequences, or introns – coding regions are called exons because they are eventually expressed in the form of a protein – RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence – the way you splice can also create multiple isoforms from one RNA transcript 5 Exon Intron Exon Pre-mRNA 5 Cap Codon 130 31104 numbers Intron Exon 3 Poly-A tail 105 146 Introns cut out and exons spliced together mRNA 5 Cap Poly-A tail 1146 5 UTR Coding segment 3 UTR • RNA splicing is carried out by spliceosomes • Spliceosomes = several proteins and small nuclear ribonucleoproteins (snRNPs) that recognize specific sequences found in introns called splice sites • snRNPs – found in the nucleus and are made of small nuclear RNA (snRNA) and proteins RNA transcript (pre-mRNA) 5 Exon 1 Protein snRNA Intron Exon 2 Other proteins snRNPs RNA transcript (pre-mRNA) 5 Exon 1 Intron Protein snRNA Exon 2 Other proteins snRNPs 1. snRNPs and other proteins combine to form the spliceosome Spliceosome 5 2. the spliceosome brings the ends of two exons together -forms a “lariat” out of the intron Spliceosome components 3. the spliceosome cuts the pre-mRNA and releases the intron for degradation 5 mRNA Exon 1 Exon 2 Cut-out intron RNA Splicing • genes can encode for more than one protein – • • DNA depending on what segments of RNA are treated as exons and what are treated as introns during splicing Exon 1 – Exon 2 Intron Exon 3 RNA processing Translation Domain 3 cut out a domain – get a different protein also - exon shuffling may result in the evolution of new proteins – Intron Transcription so the way you splice can determine what proteins eventually get made = alternative RNA splicing proteins often are composed of discrete regions called domains – coded for by distinct exons – • Gene introns increase the probability of crossingover between alleles creates new exon combinations Domain 2 Domain 1 Polypeptide Splicing • for an animation go to http://sumanasinc.com/webcontent/animatio ns/content/mRNAsplicing.html • (don’t worry about the actual proteins!) Translation • process of converting an mRNA message into a strand of amino acids that will be processed into a mature functional protein • performed by the ribosome in combination with tRNA molecules • prokaryotes - translation of mRNA can begin before transcription has finished – no separation between the mRNA and the ribosome • eukaryotic cell- the nuclear envelope separates transcription from translation – mRNA has to be exported out of the nucleus first DNA template strand 5 3 A C C A A A C C G A G T T G G T T T G G C T C A 3 5 DNA molecule Gene 1 TRANSCRIPTION Gene 2 U mRNA G G U U U G G C U C 5 A 3 Codon TRANSLATION Protein Trp Phe Gly Ser Gene 3 Amino acid • • • • – • 61 amino acid codons; 3 stop codons the code is redundant - each amino acid can be coded for by more than one codon • e.g. alanine – GCU, GCC, GCA and GCG • the GC defines the amino acid as alanine • in many cases the 3rd codon is important in defining the amino acid – serine –codons are: AGU, AGC – BUT arginine codons are: AGA and AGG The Genetic Code 1964 Second mRNA base A C U UUU Phe U UUC UAU UCU UGU Tyr UCC U UAC UGC C UCA UAA Stop UGA Stop A UUG UCG UAG Stop UGG Trp G CUU CCU CAU CGU Leu His A Cys Ser UUA C G CUC Leu CAC CCC CCA CUG CCG CAG AUU ACU AAU AUC Ile AUG CAA Met or start GUU Gln AAC ACC ACA AAA ACG AAG GCU GAU CGA C Arg CGG Asn Thr AUA CGC Pro CUA U AGU G Ser AGA U C AGC Lys A Arg A AGG G GGU U Asp G GUC GUA GUG GCC Val GCA GCG GAA GAG C GGC GAC Ala Glu GGA GGG Gly A G Third mRNA base (3 end of codon) • How are the instructions for assembling amino acids into proteins encoded into DNA? 20 amino acids - only four nucleotide bases in DNA how many nucleotides correspond to an amino acid? the mRNA nucleotide sequence is “read” in groups of 3 nucleotides = “codons” each codon codes for 1 of the 20 amino acids that make up proteins called the “genetic code” First mRNA base (5 end of codon) • Molecular Components of Translation • two components • 1. transfer RNA (tRNA) • 2. the ribosome tRNA • • • • tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long at one end – anticodon site for the hybridization with the mRNA template at the other end – attachment site for the amino acid that corresponds to the mRNA codon transcribed in the cytoplasm by RNA polymerase III – it folds into its characteristic shape spontaneously due to regions that complement each other 3 Amino acid attachment site 5 Amino acid attachment site 5 3 Hydrogen bonds Hydrogen bonds A A G 3 Anticodon (a) Two-dimensional structure Anticodon (b) Three-dimensional structure 5 Anticodon (c) Symbol used in this book Aminoacyl-tRNA synthetase (enzyme) Amino acid P Adenosine P P P Adenosine P Pi ATP Pi -amino acids are attached in the cytoplasm by enzymes called aminoacyl-tRNA –synthetases -one end fits the amino acid, the other end fits the tRNA -20 synthetases – each is specific for only one kind of tRNA -the tRNA attached to an AA is called a ‘charged tRNA’ Pi tRNA Aminoacyl-tRNA synthetase tRNA Aminoacyl tRNA (“charged tRNA”) Amino acid P Adenosine AMP Computer model tRNA and the 3rd codon “wobble” • the tRNA recognizes the codon “triplet” on the mRNA template • attached to the tRNA is the amino acid corresponding to this codon • there are 61 amino acid codons – so there should be 61 tRNAs • there are only 45 tRNAs – some tRNAs can bind more than one codon • the rules for complementary base pairing at the third NT of the codon are less stringent – “flexible” base pairing at this NT = Third Codon Wobble Ribosomes • machine of translation • made in the nucleolus in eukaryotic cells • comprised of two subunits of proteins (large and small) linked together with a piece of rRNA – eukaryotes: 40S small subunit = 33 proteins + 18S rRNA + 60S large subunit = 50 proteins + 28S rRNA (+ 5.6S rRNA + 5S rRNA) – rRNA is transcribed in the nucleolus, proteins are imported from cytoplasm – everything is assembled in the nucleolus – subunits are exported out via nuclear pores – prokaryotic ribosomes and similar but smaller Ribosomes • within the large subunit are two sites for the binding of tRNA – P-site or Peptidyl-tRNA site – “old” AA – A-site or aminoacyl-tRNA site – incoming AA • and one E site/Exit site for the exit of the tRNA off the ribosome P site (Peptidyl-tRNA binding site) Exit tunnel A site (AminoacyltRNA binding site) E site (Exit site) E mRNA binding site P A Large subunit Small subunit Ribosomes • eukaryotic ribosomes are similar but are larger vs. prokaryotes • most evidence now identifies the rRNA as being the catalyst for the formation of the peptide bond and the growth of the polypeptide chain – RNA with enzymatic activity = ribozyme Growing polypeptide Amino end Next amino acid to be added to polypeptide chain E tRNA mRNA 5 3 Codons (c) Schematic model with mRNA and tRNA Building a Polypeptide • 3 stages of translation: – Initiation – Elongation – Termination • all three stages require protein “factors” – called initiation factors or IFs – in eukaryotes – known as eIFs 1. Initiation of Translation • the small subunit of the ribosome binds onto the mRNA sequence near the 5’ methylated cap • this subunit already has an initiator tRNA (bound to methionine) associated with it • binding of the small subunit is helped by numerous eukaryotic initiation factors (eIFs) • the small subunit then glides down the mRNA “scanning” for the first codon - START codon = AUG (methionine) -stops so that initiator tRNA can hybridize with the start codon Large ribosomal subunit 3 U A C 5 5 A U G 3 P site P i Initiator tRNA mRNA GTP GDP E 5 Start codon mRNA binding site 3 Small ribosomal subunit A 5 Translation initiation complex 3 • once the small subunit is positioned - the large subunit then assembles and completes the ribosomal “machine” • helped by even more eIF’s • the mRNA and the ribsosome form the Translation Initiation Complex • the eIF’s are released once this complex forms • the ribosome is now ready for the next AA - elongation follows Large ribosomal subunit 3 U A C 5 5 A U G 3 P site P i Initiator tRNA mRNA GTP GDP E 5 Start codon mRNA binding site 3 Small ribosomal subunit A 5 Translation initiation complex 3 2. Elongation of Translation http://www.youtube.com/watc h?v=5bLEDd-PSTQ http://www.youtube.com/watc h?v=Ikq9AcBcohA http://www.youtube.com/watc h?v=NJxobgkPEAo 2. Elongation of Translation 3. Termination of Translation Release factor Free polypeptide 5 3 5 2 3 GTP 2 Stop codon (UAG, UAA, or UGA) 5 GDP 2 P -translation also stops at specific codons = STOP codons -UAA, UGA, UAG -so when the ribosome reaches these sequences – no more AAs are added and the ribosome detaches from the peptide strand and mRNA -a release factor cleaves the polypeptide chain from the tRNA and releases it from the ribosome (GTP hydrolysis) -the translation machine “breaks apart” – requires an enzyme that uses ATP hydrolysis 3 • • Polyribosomes a number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome) polyribosomes enable a cell to make many copies of a polypeptide very quickly Completed polypeptide Growing polypeptides Incoming ribosomal subunits Start of mRNA (5 end) End of mRNA (3 end) (a) Ribosomes mRNA (b) 0.1 m Mutations Mutations Wild type DNA template strand 3 T A C T T C A A A C C G A T T 5 • small-scale mutations = point mutations: • A. nucleotide pair substitutions – replacement of one NT and its partner for another pair • if there is no change to the eventual codon/amino acid = silent mutation • if it changes the amino acid to a stop codon = nonsense mutation • if it changes the amino acid = missense mutation 5 A T G A A G T T T G G C T A A 3 mRNA5 A U G A A G U U U G G C U A A 3 Protein Met Lys Phe Gly Stop Carboxyl end Amino end (a) Nucleotide-pair substitution A instead of G 3 T A C T T C A A A C C A A T T 5 5 A T G A A G T T T G G T T A A 3 U instead of C 5 A U G A A G U U U G G U U A A 3 Met Lys Phe Gly Stop Silent (no effect on amino acid sequence) T instead of C 3 T A C T T C A A A T C G A T T 5 5 A T G A A G T T T A G C T A A 3 A instead of G 5 A U G A A G U U U A G C U A A 3 Met Lys Phe Ser Stop Missense A instead of T 3 T A C A T C A A A C C G A T T 5 5 A T G T A G T T T G G C T A A 3 U instead of A 5 A U G U A G U U U G G C U A A 3 Met Nonsense Stop Mutations (b) Nucleotide-pair insertion or deletion • B. insertions • C. deletions • insertions and deletions can lead to a frame-shift • alters how the codons are read downstream from the mutation Extra A 5 3 T A C A T T C A A A C C G A T T 5 A T G T A A G T T T G G C T A A 3 A A G U U U G G C U A A 3 Extra U 5 A U G U Met Stop Frameshift causing immediate nonsense (1 nucleotide-pair insertion) missing A 3 T A 5 A T C T T G A A C A A C C G G T T G G C A T T A T 5 T A 3A U A A U missing 5 A U G A A G U Lys Met U G G Leu C 3 Ala Frameshift causing extensive missense (1 nucleotide-pair deletion) T T C missing 3 T A C A A 5 A T G T T A A 5 A U Met G G U A C C G A T T 5 T G G C T A A 3 G C U A A 3U missing U Phe U G Gly No frameshift, but one amino acid missing (3 nucleotide-pair deletion) Stop A A