Chapter 17 From Gene to Protein (Protein Synthesis) From Gene to Protein (Protein Synthesis)

Protein Synthesis  The information content of DNA  Is in the form of specific sequences of nucleotides along the DNA strands  The DNA inherited by an organism  Leads to specific traits by dictating the synthesis of proteins  Gene expression is the process by which DNA directs protein synthesis  Early experiments led researchers to: One Gene—One Polypeptide Hypothesis  The information content of DNA  Is in the form of specific sequences of nucleotides along the DNA strands  The DNA inherited by an organism  Leads to specific traits by dictating the synthesis of proteins  Gene expression is the process by which DNA directs protein synthesis  Early experiments led researchers to: One Gene—One Polypeptide Hypothesis

Protein Synthesis Overview Gene expression occurs in 2 stages: 1.Transcription  Is the synthesis of RNA under the direction of DNA  Produces messenger RNA (mRNA) Gene expression occurs in 2 stages: 1.Transcription  Is the synthesis of RNA under the direction of DNA  Produces messenger RNA (mRNA)

Protein Synthesis Overview Gene expression occurs in 2 stages: 2. Translation  Is the actual synthesis of a polypeptide, which occurs under the direction of mRNA  Occurs on ribosomes Gene expression occurs in 2 stages: 2. Translation  Is the actual synthesis of a polypeptide, which occurs under the direction of mRNA  Occurs on ribosomes

TRANSCRIPTION DNA mRNA (a) Bacterial cell TRANSLATION Ribosome Polypeptide

Protein Synthesis Overview  In eukaryotes  RNA transcripts are modified before becoming true mRNA  Initial RNA transcript is called primary transcript or pre-mRNA. Cellular chain of command  In eukaryotes  RNA transcripts are modified before becoming true mRNA  Initial RNA transcript is called primary transcript or pre-mRNA. Cellular chain of command RNARNADNADNAProteinProtein

RNA PROCESSING Nuclear envelope DNA Pre-mRNA (b) Eukaryotic cell mRNA TRANSCRIPTION TRANSLATION Ribosome Polypeptide

The Genetic Code  Genetic information  Is encoded as a sequence of non-overlapping base triplets, or codons  4 bases allow for 64 unique codons  Codons must be read in the correct reading frame  For the specified polypeptide to be produced  Genetic information  Is encoded as a sequence of non-overlapping base triplets, or codons  4 bases allow for 64 unique codons  Codons must be read in the correct reading frame  For the specified polypeptide to be produced

Different Reading Frames

Transcription & Translation  During transcription  One of the two DNA strands (template strand) provides order of complementary RNA nucleotides for mRNA transcript  Template strand always the same for a given gene  Base pairing rules apply except with uracil (U) replacing thymine (T) on the mRNA strand  During transcription  One of the two DNA strands (template strand) provides order of complementary RNA nucleotides for mRNA transcript  Template strand always the same for a given gene  Base pairing rules apply except with uracil (U) replacing thymine (T) on the mRNA strand

Transcription & Translation  During translation  mRNA codons are read in 5 ’ to 3 ’ direction  Each codon specifies 1 of the 20 amino acids for correct primary order for that polypeptide  Genetic code is redundant but not ambiguous  61 codons code for amino acids; 3 are stop codons  No codon specifies more than 1 amino acid!!!  During translation  mRNA codons are read in 5 ’ to 3 ’ direction  Each codon specifies 1 of the 20 amino acids for correct primary order for that polypeptide  Genetic code is redundant but not ambiguous  61 codons code for amino acids; 3 are stop codons  No codon specifies more than 1 amino acid!!!

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

Using the Genetic Code Overview DNA template strand TRANSCRIPTION mRNA TRANSLATION Protein Amino acid Codon Trp Phe Gly 55 55 55 55 Ser UUUUUUUUUU 33 33 33 33 55 55 33 33 GG GG GGGGCCCC TT CC AA AA AAAAAAAAAA TTTTTT TT TT GG GGGGGG CCCCCC GGGG DNA molecule Gene 1 Gene 2 Gene 3 CC CC Reading Frame

Transcription  RNA synthesis is catalyzed by RNA polymerase  Separates the DNA strands apart, hooks together the RNA nucleotides  Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine  DNA sequence where RNA polymerase attaches: Promoter  Stretch of DNA transcribed: Transcription unit  RNA synthesis is catalyzed by RNA polymerase  Separates the DNA strands apart, hooks together the RNA nucleotides  Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine  DNA sequence where RNA polymerase attaches: Promoter  Stretch of DNA transcribed: Transcription unit

Transcription  3 stages of transcription are  Initiation  Elongation  Termination  3 stages of transcription are  Initiation  Elongation  Termination

Promoter Transcription unit DNA Start point RNA polymerase Initiation 11 RNA transcript RNA transcript Unwound DNA Unwound DNA Template strand of DNA Template strand of DNA 22 Elongation Rewound DNA Rewound DNA RNA transcript RNA transcript 33 Termination Completed RNA transcript

Detailed View of Synthesis of a mRNA transcript Elongation RNA polymerase Non-template strand of DNA RNA nucleotide s 3  end C C AA UUCC CC AA A A UU TT AA GG GG TT TT A A A A C C G G U U A A T T C C AA TT CCCCAA AA T T T T G G G G 3′3′3′3′ 5′ 5′5′5′5′ Newly made RNA Direction of transcription (“downstream”) Direction of transcription (“downstream”) Template strand of DNA

Initiation  Promoters  Signal the transcriptional start point  TATA box: Important promoter in eukaryotes  Transcription factors  Mediate the binding of RNA polymerase  Transcription factors + RNA polymerase = Transcription Initiation Complex  Promoters  Signal the transcriptional start point  TATA box: Important promoter in eukaryotes  Transcription factors  Mediate the binding of RNA polymerase  Transcription factors + RNA polymerase = Transcription Initiation Complex

Initiation Transcription initiation complex forms 33 DNA Promoter Nontemplate strand 55 55 33 33 55 55 33 33 55 55 33 33 Transcription factors RNA polymerase II Transcription factors 55 55 33 33 55 55 33 33 55 55 33 33 RNA transcript Transcription initiation complex 55 55 33 33 TATA box TT TTTTTTTTTT AAAAAAAAAA AAAA TT Several transcription factors bind to DNA 22 A eukaryotic promoter 11 Start point Template strand

Elongation  RNA polymerase moves along the DNA  Untwists the double helix, 10 to 20 bases at a time  A gene can be transcribed simultaneously by several RNA polymerases  Nucleotides are added to the 3 ’ end of the growing RNA molecule  RNA polymerase moves along the DNA  Untwists the double helix, 10 to 20 bases at a time  A gene can be transcribed simultaneously by several RNA polymerases  Nucleotides are added to the 3 ’ end of the growing RNA molecule

Processing of pre-mRNA in Eukaryotes  Each end of a pre-mRNA molecule is modified in a particular way  5 ′ end receives a modified nucleotide 5 ’ cap  3 ′ end gets a poly-A tail  Each end of a pre-mRNA molecule is modified in a particular way  5 ′ end receives a modified nucleotide 5 ’ cap  3 ′ end gets a poly-A tail

Processing of pre-mRNA in Eukaryotes  These modifications share several functions  Facilitate the export of mRNA to the cytoplasm  Protect mRNA from hydrolytic enzymes  Help ribosomes attach to the 5 ’ end  These modifications share several functions  Facilitate the export of mRNA to the cytoplasm  Protect mRNA from hydrolytic enzymes  Help ribosomes attach to the 5 ’ end

RNA Processing Protein-coding segment Polyadenylation signal 55 55 33 33 33 33 55 55 55 55 Cap UTR Start codon GG PP PP PP Stop codon UTR AAUAAA Poly-A tail AAA ……

More pre-mRNA Processing  RNA splicing  Removes introns (intervening) and joins exon (expressed)  RNA splicing  Removes introns (intervening) and joins exon (expressed) TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide 5′ Cap Exon Intron Exon Intron Exon Poly-A tail Introns cut out and exons spliced together Introns cut out and exons spliced together Coding segment 5′ Cap UTR UTR Pre-mRNA mRNA

Translation  A cell translates a mRNA message into protein  With the help of transfer RNA (tRNA)  A cell translates a mRNA message into protein  With the help of transfer RNA (tRNA)

Translation  Molecules of tRNA are not all identical  Each carries a specific amino acid on one end  Each has an anticodon on the other end  Anticodons base pair with complementary codons on mRNA  Molecules of tRNA are not all identical  Each carries a specific amino acid on one end  Each has an anticodon on the other end  Anticodons base pair with complementary codons on mRNA

Structure of a tRNA molecule Amino acid attachment site 33 33 55 55 Hydrogen bonds Anticodon (a) Two-dimensional structure (b) Three-dimensional structure (c) Symbol used in book Anticodon 33 33 55 55 Hydrogen bonds Amino acid attachment site 55 55 33 33 AAAAGG

Ribosomes  Ribosomes  Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis  Ribosomes are made of 2 subunits, one large and one small  Constructed of proteins and RNA molecules named ribosomal RNA (rRNA)  Ribosomes  Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis  Ribosomes are made of 2 subunits, one large and one small  Constructed of proteins and RNA molecules named ribosomal RNA (rRNA)

Ribosomes  Ribosomes have three binding sites for tRNA  P site holds the tRNA that carries the growing polypeptide chain  A site holds the tRNA that carries the next amino acid to be added to the chain  E site is the exit site, where discharged tRNAs leave the ribosome  Ribosomes have three binding sites for tRNA  P site holds the tRNA that carries the growing polypeptide chain  A site holds the tRNA that carries the next amino acid to be added to the chain  E site is the exit site, where discharged tRNAs leave the ribosome

Ribosome Structure A site Small subunit Large subunit PP AA P site mRNA binding site E site EE

Overview of Translation TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Amino acids Amino acids tRNA with amino acid attached tRNA with amino acid attached Ribosome tRNA Anticodon mRNA Trp Phe Gly A A G G C C AA AAAA C C C C G G UU GG GGUUUUUU GGGG CC Codons

Initiation of Translation  The initiation stage of translation  Brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome  The initiation stage of translation  Brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome Large ribosomal subunit 22 mRNA mRNA binding site Small ribosomal subunit GDP GTP Start codon 11 Met UU AA CC AA UU GG EEAA

Amino end of polypeptide mRNA 55 55 EE A site 33 33 EE GTP GDP  PP ii PP AA EE PP AA GTP GDP  PP ii PP AA EE Ribosome ready for next tRNA P site

Termination of Translation  Termination occurs when a stop codon reaches the A site  A site accepts a protein called a release factor  Reaction releases the polypeptide  Termination occurs when a stop codon reaches the A site  A site accepts a protein called a release factor  Reaction releases the polypeptide

Release factor Stop codon (UAG, UAA, or UGA) 33 33 55 55 33 33 55 55 Free polypeptide 22 GTP 55 55 33 33 22 GDP  22 ii PP Termination of Translation

Mutations  Mutations  Are changes in the genetic material (DNA)  Spontaneous mutations can occur during DNA replication, recombination, or repair  Mutagens are physical or chemical agents that can cause mutations  Mutations  Are changes in the genetic material (DNA)  Spontaneous mutations can occur during DNA replication, recombination, or repair  Mutagens are physical or chemical agents that can cause mutations

Mutations  Point mutations  Are changes in just one base pair of a gene  Can be divided into two general categories  Base-pair substitutions  Base-pair insertions or deletions  Point mutations  Are changes in just one base pair of a gene  Can be divided into two general categories  Base-pair substitutions  Base-pair insertions or deletions

Mutations Summary Nucleotide-pair substitution: Replaces one nucleotide with a different one Silent mutations: Has no effect on the amino acid produced because of redundancy Missense mutations: Still codes for an amino acid, but not the correct one Nonsense mutations: Changes into a stop codon, nearly always leading to a nonfunctional protein Nucleotide-pair substitution: Replaces one nucleotide with a different one Silent mutations: Has no effect on the amino acid produced because of redundancy Missense mutations: Still codes for an amino acid, but not the correct one Nonsense mutations: Changes into a stop codon, nearly always leading to a nonfunctional protein

Wild type DNA template strand mRNA 5  55 55 Protein Amino end Stop Carboxyl end 33 33 33 33 33 33 55 55 Met Lys Phe Gly A instead of G (a) Nucleotide-pair substitution: silent Stop Met Lys Phe Gly U instead of C AA AA AAAA AAAAAAAA AAAA TT TTTTTTTTTT TTTTTTTT CCCCCCCC CC CC GGGGGGGG GG GG AA AAAAAAAA GGGGGG UUUUUUUUUU 55 55 33 33 33 33 55 55 AA AAAA AAAAAAAA AAAA TT TTTTTTTTTT TTTTTTTT CCCCCCCC GGGGGGGG AA AA AA GG AAAAAAAA GGGGGG UUUUUUUUUU TT UU 33 33 55 55

Wild type DNA template strand mRNA 5  55 55 Protein Amino end Stop Carboxyl end 33 33 33 33 33 33 55 55 Met Lys Phe Gly T instead of C (a) Nucleotide-pair substitution: missense Stop Met Lys Phe Ser A instead of G AA AA AAAA AAAAAAAA AAAA TT TTTTTTTTTT TTTTTTTT CCCCCCCC CC CC GGGGGGGG GG GG AA AAAAAAAA GGGGGG UUUUUUUUUU 55 55 33 33 33 33 55 55 AA AAAA AAAAAAAA AAAA TT TTTTTTTTTT TTTTTTTT CCCCTTCC GG GG GG AA AA GG AAAAAAAA AAGGGG UUUUUUUUUU 33 33 55 55 AACC CC GG

Wild type DNA template strand mRNA 5  55 55 Protein Amino end Stop Carboxyl end 33 33 33 33 33 33 55 55 Met Lys Phe Gly A instead of T (a) Nucleotide-pair substitution: nonsense Met AA AA AAAA AAAAAAAA AAAA TT TTTTTTTTTT TTTTTTTT CCCCCCCC CC CC GGGGGGGG GG GG AA AAAAAAAA GGGGGG UUUUUUUUUU 55 55 33 33 33 33 55 55 AA AA AAAAAAAA AAAA TT TT AA TT TTTT TT TT TT TT CCCC CC GG GG GG AA AA GG UU AAAAAA GGGG UUUUUUUUUU 33 33 55 55 CC CC GG T instead of C CC GG TT U instead of A GG Stop

Wild type DNA template strand mRNA 5  55 55 Protein Amino end Stop Carboxyl end 33 33 33 33 33 33 55 55 Met Lys Phe Gly AA AA AAAA AAAAAAAA AAAA TT TTTTTTTTTT TTTTTTTT CCCCCCCC CC CC GGGGGGGG GG GG AA AAAAAAAA GGGGGG UUUUUUUUUU (b) Nucleotide-pair insertion or deletion: frameshift causing immediate nonsense Extra A Extra U 55 55 33 33 55 55 33 33 33 33 55 55 Met 1 nucleotide-pair insertion Stop AACCAAAA GG TTTT AA TT CCTT AA CC GG TT AATTAA TT GG TT CC TT GG GG AA TT GG AA AAGG UU AA UU AAUU GG AAUU GG UUUU CC AA TT AA AA GG

DNA template strand mRNA 5  55 55 Protein Amino end Stop Carboxyl end 33 33 33 33 33 33 55 55 Met Lys Phe Gly AA AA AAAA AAAAAA AA AAAA TT TTTTTTTTTT TTTTTTTT CCCC CC CC CC CC GGGGGGGG GG GG AA AAAAAAAA GGGGGG UUUUUUUUUU (b) Codon insertion or deletion: no frameshift, but one amino acid missing Wild type AA TT CCAAAAAA AA TTTT CCCC GG TT TT CC missing Stop 55 55 33 33 33 33 55 55 33 33 55 55 Met Phe Gly 3 nucleotide-pair deletion AA GG UU CCAAAAGGGG UUUUUUUU TT GG AA AAAA TTTTTTTTCC GGGG AAAA GG

Consequences of a Point Mutation  The change of a single nucleotide in the DNA ’ s template strand  Leads to the production of an abnormal protein  The change of a single nucleotide in the DNA ’ s template strand  Leads to the production of an abnormal protein Mutant hemoglobin DNA Wild-type hemoglobin DNA mRNA Normal hemoglobin Sickle-cell hemoglobin Glu Val CC TTTT CC AATT GG AAAA GG UUAA 3′3′3′3′5′5′5′5′ ′5′5′5′3′3′3′3′5′5′5′5′3′3′3′3′