From Gene to Protein

1. Chapter 17 From Gene to Protein

2. 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

3. 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 RNA DNA Protein

4. 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

5. DNA vs. RNA

6. 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”

7. 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

8. 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

9. 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

10. 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 3’ 5’ Exon Exon Exon Exon Intron Intron Intron

11. 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

12. Transcription • genes are also associated with additional sequences of DNA 1. corepromoter 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

13. 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 pre-mRNA – 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

14. 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

15. 2 1 3 Promoter Transcription unit A eukaryotic promoter Promoter 5 3 Nontemplate strand DNA 3 5 DNA 5 3 T A A A A A T Start point 5 3 A T A T T T T RNA polymerase • Initiation – RNA polymerase binds to a special sequence • of nucleotides called the promoter TATA box Template strand Start point Several transcriptionfactors bind to DNA Transcriptionfactors -certain sections of the promoter are important in polymerase binding = core promoter -in prokaryotes the promoter binds the RNA polymerase without help -in eukaryotes – the polymerase requires the assistance of proteins called transcription factors -specific transcription factors bind to the promoter first and then help position the polymerase at the promoter -additional transcription factors then bind -entire complex is called the Transcription Initiation Complex 5 3 3 5 Transcription initiationcomplex forms RNA polymerase II Transcription factors 5 3 3 5 3 5 RNA transcript Transcription initiation complex sequence given in texts is that of the sense strand

16. 1 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase Initiation Nontemplate strand of DNA 3 5 5 3 Template strand of DNA RNAtranscript UnwoundDNA • 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

17. 1 2 Promoter Nontemplatestrand of DNA Transcription unit 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 5 3 RNA nucleotides 3 5 DNA Start point RNApolymerase RNA polymerase Initiation Nontemplate strand of DNA 3 5 C C A A T A 5 3 5 T 3 Template strand of DNA U RNAtranscript T C UnwoundDNA end 3 G T Elongation U A G C A RewoundDNA C C U C A A C A 3 5 3 5 A 3 5 3 5 T A G G T T RNAtranscript 5 Direction of transcription Templatestrand of DNA Multiple RNA polymerases per DNA template Newly madeRNA

18. 3 1 2 Promoter Transcription unit 5 3 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 poly-adenylation signal – for the release of the pre-RNA from the polymerase 3 5 DNA Start point RNA polymerase Initiation Nontemplate strand of DNA 3 5 5 3 Template strand of DNA RNAtranscript UnwoundDNA Elongation RewoundDNA 3 5 3 5 3 5 RNAtranscript Termination 3 5 5 3 3 5 Completed RNA transcript Direction of transcription (“downstream”)

19. 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

20. 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 Protein-codingsegment Polyadenylationsignal 5 3 … G P P P AAA AAUAAA AAA Startcodon Stopcodon Cap 5 UTR 5 Poly-A tail 3 UTR

21. 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 Protein-codingsegment Polyadenylationsignal 5 3 … G P P P AAA AAUAAA AAA Startcodon Stopcodon Cap 5 UTR 5 Poly-A tail 3 UTR

22. 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 Exon Intron Exon Intron Exon 5 3 Poly-A tail Pre-mRNACodonnumbers Cap 5 130 31104 105 146 Introns cut out andexons spliced together 5 mRNA Cap Poly-A tail 1146 UTR 3 UTR 5 Codingsegment

23. 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 Intron Exon 2 Protein Other proteins snRNA snRNPs

24. RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs • 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 Spliceosomecomponents Cut-outintron 3. the spliceosome cuts the pre-mRNA and releases the intron for degradation mRNA 5 Exon 1 Exon 2

25. RNA Splicing genes can encode for more than one protein depending on what segments of RNA are treated as exons and what are treated as introns during splicing 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 cut out a domain – get a different protein also - exon shuffling may result in the evolution of new proteins introns increase the probability of crossing-over between alleles creates new exon combinations Gene DNA Intron Exon 2 Intron Exon 3 Exon 1 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide

26. Splicing • for an animation go to http://sumanasinc.com/webcontent/animations/content/mRNAsplicing.html • (don’t worry about the actual proteins!)

27. Translation DNAtemplatestrand DNA 5 3 molecule A A A A A C C C C T G G • 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 T T T T A G G G G C T C Gene 1 3 5 TRANSCRIPTION Gene 2 U G G U U U G G C U C A 5 3 mRNA Codon TRANSLATION Gly Phe Trp Protein Ser Gene 3 Amino acid

28. The Genetic Code Second mRNA base A U C G UUU UAU UCU UGU U Phe Cys Tyr 1964 UUC UCC UAC UGC C U Ser UUA UCA UGA Stop A UAA Stop Leu • 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” • 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 Trp UUG UCG UGG G UAG Stop CUU CCU U CAU CGU His CUC CAC CGC C CCC C Leu Pro Arg CUA CCA CGA A CAA Gln CUG CCG CGG G CAG First mRNA base (5 end of codon) Third mRNA base (3 end of codon) AUU AAU ACU AGU U Ser Asn C AUC Ile AAC ACC AGC A Thr AUA AAA ACA AGA A Lys Arg Met orstart AUG ACG AGG AAG G GUU GCU GAU GGU U Asp GUC GCC C GGC GAC G Val Ala Gly Gly GUA GCA GGA A GAA Glu GUG GCG GGG G GAG

29. Molecular Components of Translation two components 1. transfer RNA (tRNA) 2. the ribosome

30. tRNA 3 • 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 Amino acidattachmentsite 5 Amino acidattachmentsite 5 3 Hydrogenbonds Hydrogenbonds A A G 3 5 Anticodon Anticodon Anticodon (c) Symbol used (a) Two-dimensional structure in this book (b) Three-dimensional structure

31. Aminoacyl-tRNAsynthetase (enzyme) Amino acid P Adenosine P P P Adenosine P P i Aminoacyl-tRNAsynthetase ATP P tRNA i P i -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’ tRNA Aminoacid P Adenosine AMP Computer model Aminoacyl tRNA(“charged tRNA”)

32. 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

33. 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

34. A site (Aminoacyl-tRNA binding site) Ribosomes P site (Peptidyl-tRNAbinding site) Exit tunnel • 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 E site (Exit site) E A P Largesubunit mRNAbinding site Smallsubunit

35. Ribosomes Growing polypeptide Amino end Next aminoacid to beadded topolypeptidechain • 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 E tRNA mRNA 3 Codons 5 (c) Schematic model with mRNA and tRNA

36. 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

37. 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 Largeribosomalsubunit U 3 5 C A P site Met Met 3 5 A G U P i InitiatortRNA  GTP GDP E A mRNA 5 5 3 3 Start codon Smallribosomalsubunit Translation initiation complex mRNA binding site

38. 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 Largeribosomalsubunit U 3 5 C A P site Met Met 3 5 A G U P i InitiatortRNA  GTP GDP E A mRNA 5 5 3 3 Start codon Smallribosomalsubunit Translation initiation complex mRNA binding site

39. 2. Elongation of Translation http://www.youtube.com/watch?v=5bLEDd-PSTQ http://www.youtube.com/watch?v=Ikq9AcBcohA http://www.youtube.com/watch?v=NJxobgkPEAo

40. 2. Elongation of Translation

41. P 3. Termination of Translation Releasefactor Freepolypeptide 5 3 3 3 GTP 2 5 5  2 GDP 2 Stop codon (UAG, UAA, or UGA) -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

42. 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 Completedpolypeptide Growingpolypeptides Incomingribosomalsubunits Polyribosome Start ofmRNA(5 end) End ofmRNA(3 end) (a) Ribosomes mRNA (b) 0.1 m