IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: C. RNA Processing: D. Deciphering the Genetic Code

IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: C. RNA Processing: D. Deciphering the Genetic Code 1. Sidney Brenner – suggested a triplet code (minimum necessary to encode 20 AA)

IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: C. RNA Processing: D. Deciphering the Genetic Code 1. Sidney Brenner – suggested a triplet code (minimum necessary to encode 20 AA) 2. Crick analyzed addition/deletion mutations, and confirmed a triplet code that is non- overlapping.

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 3. Nirenberg and Mattaei – 1961: Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA bases – mRNA.

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 3. Nirenberg and Mattaei – 1961: Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA bases – mRNA. Then added t-RNA’s, ribosomes, and amino acids, the chemical reactions would make protein based on this m-RNA sequence. (in vitro) polypeptide

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 3. Nirenberg and Mattaei – 1961: Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA bases – mRNA. Then added t-RNA’s, ribosomes, and amino acids, the chemical reactions would make protein based on this m-RNA sequence. (in vitro) Then they could isolate and digest the protein and see which AA’s had been incorporated, and at what fractions…. 60%40%

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 3. Nirenberg and Mattaei – 1961: Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA bases – mRNA. Then added t-RNA’s, ribosomes, and amino acids, the chemical reactions would make protein based on this m-RNA sequence. (in vitro) Then they could isolate and digest the protein and see which AA’s had been incorporated, and at what fractions…. Homopolymers were easy: make UUUUUUU RNA, get polypeptide with only phenylalanine

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 3. Nirenberg and Mattaei – 1961: Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA bases – mRNA. Then added t-RNA’s, ribosomes, and amino acids, the chemical reactions would make protein based on this m-RNA sequence. (in vitro) Then they could isolate and digest the protein and see which AA’s had been incorporated, and at what fractions…. Homopolymers were easy: make UUUUUUU RNA, get polypeptide with only phenylalanine make AAAAAAA RNA, get polypeptide with only lysine make CCCCCCCC RNA, get polypeptide with only proline make GGGGGGG RNA, and the molecule folds back on itself… (oh well).

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 3. Nirenberg and Mattaei – 1961: Homopolymers were easy: Heteropolymers were more clever: add two bases at different ratios (1/6 A, 5/6 C):

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 3. Nirenberg and Mattaei – 1961: Homopolymers were easy: Heteropolymers were more clever: add two bases at different ratios (1/6 A, 5/6 C): So, since the enzyme links bases randomly (there is no template), you can predict how frequent certain 3-base combinations should be: AAA = 1/6 x 1/6 x 1/6 = 1/216 = 0.4%

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 3. Nirenberg and Mattaei – 1961: figured out 50 of the 64 codons 4. Khorana Dinucleotide, trinucleotides, and tetranucleotides: make specific triplets He confirmed existing triplets, filled in others, and identified stop codons because of premature termination. Nobels for Nirenberg and Khorana!!

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 5. Patterns The third position is often not critical, such that U at the first position of the t-RNA (its antiparallel) can pair with either A or G in the m-RNA. This reduces the number of t-RNA molecules needed. 5’3’ M-RNA C G C A U A C A C A A 5’3’ U G U

IX: DNA Function: Protein Synthesis D. Deciphering the Code: 5. Patterns The third position is often not critical, such that U at the first position of the t- RNA (its antiparallel) can pair with either A or G in the m-RNA. This reduces the number of t-RNA molecules needed. There are also some chemical similarities to the amino acids encoded by similar codons, which may have persisted as the code evolved because errors were not as problematic to protein function.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription a. The message is on one strand of the double helix - the sense strand: 3’ 5’ In all eukaryotic genes and in some prokaryotic sequences, there are introns and exons. There may be multiple introns of varying length in a gene. Genes may be several thousand base-pairs long. This is a simplified example! A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A sense nonsense intronexon

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription b. The cell 'reads' the correct strand based on the location of the promoter, the anti- parallel nature of the double helix, and the chemical limitations of the 'reading' enzyme, RNA Polymerase. 3’ 5’ Promoters have sequences recognized by the RNA Polymerase. They bind in particular orientation. A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A sense nonsense intronexon Promoter

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription b. The cell 'reads' the correct strand based on the location of the promoter, the anti- parallel nature of the double helix, and the chemical limitations of the 'reading' enzyme, RNA Polymerase. 3’ 5’ 1)Strand separate 2)RNA Polymerase can only synthesize RNA in a 5’  3’ direction, so they only read the anti-parallel, 3’  5’ strand (‘sense’ strand). A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A sense nonsense intronexon Promoter G C A U GUUU G C C A A U AUG A U G A

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription c. Transcription ends at a sequence called the 'terminator'. Terminator sequences destabilize the RNA Polymerase and the enzyme decouples from the DNA, ending transcription 3’ 5’ A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A sense nonsense intronexon Promoter G C A U GUUU G C C A A U AUG A U G A Terminator

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription c. Transcription ends at a sequence called the 'terminator'. 3’ 5’ A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A sense nonsense intronexon Promoter G C A U GUUU G C C A A U AUG A U G A Terminator Initial RNA PRODUCT:

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription c. Transcription ends at a sequence called the 'terminator'. 3’ 5’ A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A sense nonsense PromoterTerminator intronexon G C A U GUUU G C C A A U AUG A U G A Initial RNA PRODUCT:

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing intronexon Initial RNA PRODUCT: Introns are spliced out, and exons are spliced together. Sometimes these reactions are catalyzed by the intron, itself, or other catalytic RNA molecules called “ribozymes”. G C A U GUUU G C C A A UAUG AU G A

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing intron exon Final RNA PRODUCT: This final RNA may be complexed with proteins to form a ribosome (if it is r-RNA), or it may bind amino acids (if it is t-RNA), or it may be read by a ribosome, if it is m- RNA and a recipe for a protein. G C A U GUUU G C C A A U AUG A U G A

IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: C. RNA Processing: D. Deciphering the Code: E. Translation!!! 1. Players: a. processed m-RNA transcript: binding site (Shine-Delgarno sequence in bacteria: AGGAGG) (Kazak sequence in eukaryotes: ACCAUGG)

IX: DNA Function: Protein Synthesis A. Overview: B. Deciphering the Code: C. Transcription: D. RNA Processing: E. Translation!!! 1. Players: a. processed m-RNA transcript: binding site (Shine-Delgarno sequence in bacteria: AGGAGG) (Kazak sequence in eukaryotes: ACCAUGG) start codon (AUG)

IX: DNA Function: Protein Synthesis A. Overview: B. Deciphering the Code: C. Transcription: D. RNA Processing: E. Translation!!! 1. Players: a. processed m-RNA transcript: binding site (Shine-Delgarno sequence in bacteria: AGGAGG) (Kazak sequence in eukaryotes: ACCAUGG) start codon (AUG) codon sequence….

IX: DNA Function: Protein Synthesis A. Overview: B. Deciphering the Code: C. Transcription: D. RNA Processing: E. Translation!!! 1. Players: a. processed m-RNA transcript: binding site (Shine-Delgarno sequence in bacteria: AGGAGG) (Kazak sequence in eukaryotes: ACCAUGG) start codon (AUG) codon sequence…. stop codon (UGA, etc…)

IX: DNA Function: Protein Synthesis A. Overview: B. Deciphering the Code: C. Transcription: D. RNA Processing: E. Translation!!! 1. Players: a. processed m-RNA transcript: binding site (Shine-Delgarno sequence in bacteria: AGGAGG) (Kazak sequence in eukaryotes: ACCAUGG) start codon (AUG) codon sequence…. stop codon (UGA, etc…) 7mG cap and poly-A tail in eukaryotes

IX: DNA Function: Protein Synthesis A. Overview: B. Deciphering the Code: C. Transcription: D. RNA Processing: E. Translation!!! 1. Players: a. processed m-RNA transcript: b. Ribosome: 2 subunits (large and small) each with a peptidyl site (P) and aminoacyl site (A).

IX: DNA Function: Protein Synthesis A. Overview: B. Deciphering the Code: C. Transcription: D. RNA Processing: E. Translation!!! 1. Players: a. processed m-RNA transcript: b. Ribosome c. T-RNA and AA’s

E. Translation!!! 1. Players: a. processed m-RNA transcript: b. Ribosome c. T-RNA and AA’s d. Protein factors – increase efficiency of process

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s Each t-RNA is bound to a specific AA by a very specific enzyme; a unique form of aminoacyl synthetase. The specificity of each enzyme is responsible for the unambiguous genetic code.

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: - METH-t-RNA binds to SRS in p-site, forming the Initiation Complex

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: -METH-t-RNA binds to SRS in p-site, forming the Initiation Complex -The LRS binds to this complex, completing th aminoacyl site – the first base is in position and we are ready to polymerize…

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: c. Elongation (Polymerization): -The second AA-t-RNA complex binds in the Acyl site.

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: c. Elongation (Polymerization): -The second AA-t-RNA complex binds in the Acyl site. -Translocation reaction: - Peptidyl transferase makes a Peptide bond between the adjacent AA’s.

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: c. Elongation (Polymerization): -The second AA-t-RNA complex binds in the Acyl site. -Translocation reaction: - Peptidyl transferase makes a Peptide bond between the adjacent AA’s. - Uncharged t-RNA shifts to e-site And is released from ribosome, while the m- RNA, t-RNA complex shifts to the p-site…

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: c. Elongation (Polymerization): -The second AA-t-RNA complex binds in the Acyl site. -Translocation reaction: - Peptidyl transferase makes a Peptide bond between the adjacent AA’s. - Uncharged t-RNA shifts to e-site And is released from ribosome, while the m- RNA, t-RNA complex shifts to the p-site… - the A-site is now open and across From the next m-RNA codon; ready to accept The next charged t-RNA

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: c. Elongation (Polymerization): -The second AA-t-RNA complex binds in the Acyl site. -Translocation reaction - The third charged t-RNA enters the A-site

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: c. Elongation (Polymerization): -And another translocation reaction occurs

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: c. Elongation (Polymerization): -And another translocation reaction occurs…. This is repeated until….

E. Translation!!! 1. Players: 2. Process: a. Charging t-RNA’s b. Initiation: c. Elongation (Polymerization): d. Termination: When a stop codon is reached (not the last codon, as shown in the picture…), no charged t-RNA is placed in the A-site… this signals GTP-releasing factors to cleave the polypeptide from the t-RNA, releasing it from the ribosome.

E. Translation!!! 1. Players: 2. Process: 3. Polysomes: M-RNA’s last for only minutes or hours before their bases are cleaved and recycled. Productivity is amplified by having multiple ribosomes reading down the same m- RNA molecule; creating the ‘polysome’ structure seen here.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. M-RNA:G C A U G U U U G C C A A UU G A

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. M-RNA:G C A U G U U U G C C A A UU G A It then reads down the m-RNA, one base at a time, until an ‘AUG’ sequence (start codon) is positioned in the first reactive site.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds to the m-RNA/ribosome complex. M-RNA:G C A U G U U U G C C A A UU G A Meth

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds to the m-RNA/ribosome complex. c. A second t-RNA-AA binds to the second site M-RNA:G C A U G U U U G C C A A UU G A MethPhe

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds to the m-RNA/ribosome complex. c. A second t-RNA-AA binds to the second site d. Translocation reactions occur M-RNA:G C A U G U U U G C C A A UU G A Meth Phe The amino acids are bound and the ribosome moves 3-bases “downstream”

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation e. polymerization proceeds M-RNA:G C A U G U U U G C C A A UU G A MethPhe The amino acids are bound and the ribosome moves 3-bases “downstream” AlaAsn

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation e. polymerization proceeds M-RNA:G C A U G U U U G C C A A UU G A MethPhe The amino acids are bound and the ribosome moves 3-bases “downstream” Ala Asn

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation e. polymerization proceeds f. termination of translation M-RNA:G C A U G U U U G C C A A UU G A Some 3-base codon have no corresponding t-RNA. These are stop codons, because translocation does not add an amino acid; rather, it ends the chain. MethPheAlaAsn

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation 4. Post-Translational Modifications Most initial proteins need to be modified to be functional. Most need to have the methionine cleaved off; others have sugar, lipids, nucleic acids, or other proteins are added. MethPheAlaAsn

IX: DNA Function: Protein Synthesis A. Overview: B. Deciphering the Code: C. Transcription: D. RNA Processing: E. Translation!!! - Summary: The nucleotide sequence in DNA determines the amino acid sequence in proteins. A single change in that DNA sequence can affect a single amino acid, and may affect the structure and function of that protein.

IX: DNA Function: Protein Synthesis A. Overview: B. Deciphering the Code: C. Transcription: D. RNA Processing: E. Translation!!! - Summary: The nucleotide sequence in DNA determines the amino acid sequence in proteins. A single change in that DNA sequence can affect a single amino acid, and may affect the structure and function of that protein. Because all biological processes are catalyzed by either RNA or protienaceous enzymes, and because proteins are also primary structural, transport, and immunological molecules in living cells, changes in protein structure can change how living systems work. Evolution occurs through changes in DNA, which cause changes in proteins and affect how and when they act in living cells.

IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. The code is: - linear

IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. The code is: - linear - ‘triplet’ Three DNA/RNA bases are a ‘word’ that specifies a single amino acid. This is the minimum number need to specific the 20 AA’s found in living systems.

IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. The code is: - linear - ‘triplet’ - ‘unambiguous’ Each three-base sequence (RNA ‘codon’) codes for only ONE amino acid.

IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. The code is: - linear - ‘triplet’ - ‘unambiguous’ - ‘degenerate’ (redundant) Each amino acid can be coded for by more than one three-base codon.

IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. The code is: - linear - ‘triplet’ - ‘unambiguous’ - ‘degenerate’ (redundant) - ‘start and stop signals’ There are specific codons that signal translation enzymes where to start and stop.

IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. The code is: - linear - ‘triplet’ - ‘unambiguous’ - ‘degenerate’ (redundant) - ‘start and stop signals’ - ‘commaless’ There is no internal punctuation; translation proceeds from start signal to stop signal.

IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. The code is: - linear - ‘triplet’ - ‘unambiguous’ - ‘degenerate’ (redundant) - ‘start and stop signals’ - ‘commaless’ - ‘non-overlapping’ AACGUA is read: ‘AAC’ ‘GUA’ not: ‘AAC’ ‘ACG’ ‘CGU’ ‘GUA’

IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. The code is: - linear - ‘triplet’ - ‘unambiguous’ - ‘degenerate’ (redundant) - ‘start and stop signals’ - ‘commaless’ - ‘non-overlapping’ - ‘universal’ With rare exceptions in single codons, all life forms use the exact same ‘dictionary’… so AAA codes for lysine in all life. There is one language of life, suggesting a single origin.