Protein Synthesis

The Flow of Genetic Information The information carried by DNA is in the form of specific sequences of nucleotides Your DNA is a set of instructions that your cells follow to make proteins. The proteins you have determine your traits. Gene expression, the process by which DNA directs protein synthesis, has two stages: transcription and translation Basic process: DNA  mRNA  Protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Basic Principles of Transcription and Translation In transcription, a piece of DNA that codes for a specific protein is copied onto messenger RNA (mRNA). This mRNA can move out of the nucleus to the place where the protein is needed, and if anything happens to it, the DNA will remain undamaged in the nucleus. In translation, a ribosome reads the instructions off of the mRNA copy and puts a protein together from specific amino acids, based on the mRNA instructions. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Types of RNA In all, there are 3 types of RNA involved in the transcription/translation process mRNA (messenger RNA; RNA built in the nucleus that travels to the cytoplasm) rRNA (ribosomal RNA; makes up ribosomes that the mRNA is translated on) tRNA (transfer RNA; translates mRNA into an amino acid, and thus a polypeptide chain or protein)

In a eukaryotic cell, the nuclear envelope separates transcription from translation Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Nuclear envelope DNA TRANSCRIPTION Pre-mRNA (b) Eukaryotic cell Fig. 17-3b-1 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information (b) Eukaryotic cell

Nuclear envelope DNA TRANSCRIPTION Pre-mRNA mRNA (b) Eukaryotic cell Fig. 17-3b-2 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information (b) Eukaryotic cell

Nuclear envelope DNA TRANSCRIPTION Pre-mRNA mRNA TRANSLATION Ribosome Fig. 17-3b-3 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

Summary of Transcription and Translation DNA  mRNA (happens in the nucleus) Translation mRNA  polypeptide (happens in the cytoplasm) - Ribosomes help

The Genetic Code How are the instructions for assembling amino acids into proteins encoded into DNA? There are 20 amino acids, but there are only four nucleotide bases in DNA How many bases correspond to an amino acid? 3 (next slide) Remember – Proteins are strings of amino acid monomers put together Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Codons: Triplets of Bases Every 3 consecutive nucleotides represent a triplet, which your ribosomes read like a word. Each 3-letter “word” on the DNA codes for a specific amino acid Example: AGT on the DNA tells the ribosome to add the amino acid Serine Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

During transcription, mRNA nucleotides attach to one of the two DNA strands (called the template strand). During translation, the mRNA triplets (the 3-letter words), called codons, are read in the 5 to 3 direction (the letter at the 5’ end is at the beginning of the codon word). Each codon specifies which amino acid should be added to the polypeptide next Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Gene 2 Gene 1 Gene 3 DNA template strand mRNA Codon TRANSLATION Fig. 17-4 Gene 2 DNA molecule Gene 1 Gene 3 DNA template strand TRANSCRIPTION Figure 17.4 The triplet code mRNA Codon TRANSLATION Protein Amino acid

Cracking the Code All 64 codons were deciphered by the mid-1960s Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation Just as some words mean the same thing, multiple codons can give the same amino acid Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Reading frames For the sequence ‘AUGCCGAC’, you could start at the beginning and read the codons “AUG, CCG” If you used a different reading frame, you could read “UGC, CGA” or “GCC, GAC” If you start in the wrong reading frame, you’ll combine the wrong amino acids to make the wrong protein. Since AUG is the start codon, ribosomes look for an AUG and start reading there.

Start and Stop Codons Start AUG Stop UAA UAG UGA

First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Fig. 17-5 Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Figure 17.5 The dictionary of the genetic code

Evolution of the Genetic Code The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals Genes can be transcribed and translated after being transplanted from one species to another The same sequence of nitrogenous bases in the DNA will code for the same amino acids and proteins in almost all species. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

(a) Tobacco plant expressing a firefly gene (b) Pig expressing a Fig. 17-6 Figure 17.6 Expression of genes from different species (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene

Molecular Components of Transcription RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine RNA has U instead of T Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Nontemplate Elongation strand of DNA RNA nucleotides RNA polymerase 3 Fig. 17-7b Elongation Nontemplate strand of DNA RNA nucleotides RNA polymerase 3 3 end 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 Direction of transcription (“downstream”) Template strand of DNA Newly made RNA

The stretch of DNA that is transcribed is called a transcription unit The DNA sequence where RNA polymerase first attaches is called the promoter A promoter is necessary to initiate RNA transcription The stretch of DNA that is transcribed is called a transcription unit Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase Fig. 17-7a-1 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase Figure 17.7 The stages of transcription: initiation, elongation, and termination

Elongation of the RNA Strand As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 40 nucleotides per second in eukaryotes A gene can be transcribed by several RNA polymerases at a time Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase Fig. 17-7a-2 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation 5 3 3 5 RNA transcript Template strand of DNA Unwound DNA Figure 17.7 The stages of transcription: initiation, elongation, and termination

Nontemplate Elongation strand of DNA RNA nucleotides RNA polymerase 3 Fig. 17-7b Elongation Nontemplate strand of DNA RNA nucleotides RNA polymerase 3 3 end 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 Direction of transcription (“downstream”) Template strand of DNA Newly made RNA

Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase Fig. 17-7a-3 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation 5 3 3 5 RNA transcript Template strand of DNA Unwound DNA 2 Elongation Rewound DNA 5 3 3 3 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 RNA transcript

Termination of Transcription In eukaryotes, the polymerase continues transcription and eventually falls off the DNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Completed RNA transcript Fig. 17-7a-4 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation 5 3 3 5 RNA transcript Template strand of DNA Unwound DNA 2 Elongation Rewound DNA 5 3 3 3 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 RNA transcript 3 Termination 5 3 3 5 5 3 Completed RNA transcript

Eukaryotic cells modify RNA after transcription During RNA processing, both ends of the primary transcript are usually altered Also, usually some interior parts of the molecule are cut out, and the other parts spliced together Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Alteration of mRNA Ends Each end of a pre-mRNA molecule is modified in a particular way: The 5 end receives a modified nucleotide 5 cap The 3 end gets a poly-A tail Why add these ends? They seem to make it easier to move mRNA out of the nucleus They protect mRNA from hydrolytic (causing hydrolysis) enzymes They help ribosomes attach to the 5 end Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Protein-coding segment PolyA signal 5 3 Fig. 17-9 Protein-coding segment PolyA signal 5 3 G P P P AAUAAA AAA … AAA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail Figure 17.9 RNA processing: addition of the 5 cap and poly-A tail

Split Genes and RNA Splicing Most eukaryotic genes and their RNA transcripts have pieces that don’t code for useful proteins. These noncoding regions are called introns The other parts are called exons because they are eventually expressed (translated into amino acid sequences) RNA splicing removes the introns and joins the exons together, creating mRNA with a continuous coding sequence Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

exons spliced together Coding segment Fig. 17-10 5 Exon Intron Exon Intron Exon 3 Pre-mRNA 5 Cap Poly-A tail 1 30 31 104 105 146 Introns cut out and exons spliced together Coding segment mRNA 5 Cap Poly-A tail 1 146 Figure 17.10 RNA processing: RNA splicing

The Functional and Evolutionary Importance of Introns Some genes can code for more than one kind of protein, depending on which segments are treated as exons during RNA splicing These variations are called alternative RNA splicing Because of alternative RNA splicing, the number of different proteins an organism can produce is much greater than its number of genes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

After RNA processing, the mRNA is translated. A cell translates an mRNA message into protein with the help of transfer RNA (tRNA) Molecules of tRNA are not identical: Each carries a specific amino acid on one end Each has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Amino acids tRNA with amino acid attached Ribosome tRNA Anticodon 5 Fig. 17-13 Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly Figure 17.13 Translation: the basic concept tRNA Anticodon 5 Codons 3 mRNA

Accurate translation requires two steps: First: a correct match between a tRNA and an amino acid (tRNA with an amino acid attached is “charged” tRNA) Second: a correct match between the tRNA anticodon and an mRNA codon Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Ribosomes Ribosomes help tRNA anticodons to pair up with specific mRNA codons in protein synthesis The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

(b) Schematic model showing binding sites Fig. 17-16b P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) E P A Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites Growing polypeptide Amino end Next amino acid to be added to polypeptide chain Figure 17.16 The anatomy of a functioning ribosome E tRNA mRNA 3 Codons 5 (c) Schematic model with mRNA and tRNA

Initiation of Translation First, a small ribosomal subunit binds with mRNA and a special initiator tRNA Then the small subunit moves along the mRNA until it reaches the start codon (AUG) Proteins called initiation factors bring in the large subunit If the initiator tRNA sticks to the AUG codon, what is the anticodon on this tRNA? Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Translation initiation complex Fig. 17-17 Large ribosomal subunit 3 U C 5 A P site Met 5 A Met U G 3 Initiator tRNA GTP GDP E A mRNA 5 5 3 3 Start codon Figure 17.17 The initiation of translation Small ribosomal subunit mRNA binding site Translation initiation complex

Amino end of polypeptide E 3 mRNA 5 Fig. 17-18-1 P A site site Figure 17.18 The elongation cycle of translation

GDP Amino end of polypeptide E 3 mRNA 5 E P A Fig. 17-18-2 P A site GTP GDP E P A Figure 17.18 The elongation cycle of translation

GDP Amino end of polypeptide E 3 mRNA 5 E P A E P A Fig. 17-18-3 P A site A site 5 GTP GDP E P A Figure 17.18 The elongation cycle of translation E P A

GDP GDP Amino end of polypeptide E 3 mRNA Ribosome ready for Fig. 17-18-4 Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P site A site 5 GTP GDP E E P A P A Figure 17.18 The elongation cycle of translation GDP GTP E P A

Termination of Translation Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome The A site accepts a protein called a release factor The release factor adds a water molecule instead of an amino acid The polypeptide, the mRNA, and the two ribosomal subunits come apart and float off into the cytoplasm Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Release factor 3 5 Stop codon (UAG, UAA, or UGA) Fig. 17-19-1 Figure 17.19 The termination of translation

Release factor Free polypeptide 3 3 2 5 5 Stop codon Fig. 17-19-2 Release factor Free polypeptide 3 3 2 5 5 GTP Stop codon (UAG, UAA, or UGA) 2 GDP Figure 17.19 The termination of translation

Release factor Free polypeptide 5 3 3 3 2 5 5 Stop codon Fig. 17-19-3 Release factor Free polypeptide 5 3 3 3 2 5 5 GTP Stop codon (UAG, UAA, or UGA) 2 GDP Figure 17.19 The termination of translation

Polyribosomes A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome Polyribosomes enable a cell to make many copies of a polypeptide very quickly Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Completed polypeptide Growing polypeptides Incoming ribosomal subunits Fig. 17-20 Completed polypeptide Growing polypeptides Incoming ribosomal subunits Polyribosome Start of mRNA (5 end) End of mRNA (3 end) (a) Ribosomes Figure 17.20 Polyribosomes mRNA (b) 0.1 µm

Targeting Polypeptides to Specific Locations Cells have 2 kinds of ribosomes: free ribosomes and bound ribosomes Free ribosomes mostly synthesize proteins that are used in the cytoplasm Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell Ribosomes are identical and can switch from free to bound Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Protein synthesis always begins in the cytoplasm It finishes in the cytoplasm unless the polypeptide signals the ribosome to attach to the ER Polypeptides destined for the ER are marked by a signal peptide Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Types of Mutations Mutations are changes in the genetic material of a cell or virus Point mutations are changes in just one base pair of a gene The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Wild-type hemoglobin DNA Mutant hemoglobin DNA 3 C T T 5 3 C A T 5 Fig. 17-22 Wild-type hemoglobin DNA Mutant hemoglobin DNA 3 C T T 5 3 C A T 5 5 G A A 3 5 G T A 3 mRNA mRNA 5 G A A 3 5 G U A 3 Figure 17.22 The molecular basis of sickle-cell disease: a point mutation Normal hemoglobin Sickle-cell hemoglobin Glu Val

Types of Point Mutations Point mutations within a gene can be divided into two general categories Base-pair substitutions Base-pair insertions or deletions Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Substitutions A base-pair substitution replaces one nucleotide and its partner with another pair of nucleotides Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code Missense mutations still code for an amino acid, but not the amino acid is different Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Silent (no effect on amino acid sequence) Fig. 17-23a Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of G 3 5 5 3 Figure 17.23 Types of point mutations U instead of C 5 3 Stop Silent (no effect on amino acid sequence)

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Fig. 17-23b Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end T instead of C 3 5 5 3 Figure 17.23 Types of point mutations A instead of G 5 3 Stop Missense

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Fig. 17-23c Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of T 3 5 5 3 Figure 17.23 Types of point mutations U instead of A 5 3 Stop Nonsense

Insertions and Deletions Insertions and deletions are additions or losses of nucleotide pairs in a gene Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation These mutations can change the protein much more than a substitution does, and can be disastrous Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Frameshift causing immediate nonsense (1 base-pair insertion) Fig. 17-23d Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end Extra A 3 5 5 3 Figure 17.23 Types of point mutations Extra U 5 3 Stop Frameshift causing immediate nonsense (1 base-pair insertion)

Frameshift causing extensive missense (1 base-pair deletion) Fig. 17-23e Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 3 5 5 3 Figure 17.23 Types of point mutations missing 5 3 Frameshift causing extensive missense (1 base-pair deletion)

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Fig. 17-23f Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 3 5 5 3 Figure 17.23 Types of point mutations missing 5 3 Stop No frameshift, but one amino acid missing (3 base-pair deletion)

What Is a Gene? Revisiting the Question The idea of the gene itself is a unifying concept of life We have considered a gene as: A discrete unit of inheritance A region of specific nucleotide sequence in a chromosome A DNA sequence that codes for a specific polypeptide chain Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-25 DNA TRANSCRIPTION 3 Poly-A RNA polymerase 5 RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Poly-A NUCLEUS Amino acid AMINO ACID ACTIVATION CYTOPLASM tRNA mRNA Growing polypeptide Cap 3 A Activated amino acid Poly-A P Ribosomal subunits Figure 17.25 A summary of transcription and translation in a eukaryotic cell E Cap 5 TRANSLATION E A Anticodon Codon Ribosome

In summary, a gene can be defined as a piece of DNA that can be expressed to produce a functional protein and is inherited by organisms from their parents Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings