Chapter 17 RNA and Protein Synthesis

Transcription and Translation http://www.dnatube.com/video/3059/DNA-Transcription-and-Protein-Assembly

Overview Nucleotide sequences responsible for information on DNA The DNA inherited yields specific traits by dictating the synthesis of proteins Proteins are the links between genotype and phenotype Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation

History of Gene Expression 1909 – Archibald Garrod First to say genes determine phenotypes through enzymes that catalyze specific chemical reactions “inborn errors of metabolism” - symptoms of an inherited disease reflect an inability to synthesize a certain enzyme

History of Gene Expression George Beadle and Edward Tatum exposed bread mold to X-rays mutants were unable to survive on minimal medium – unable to synthesize certain molecules Identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine One gene–one enzyme hypothesis: states that each gene dictates production of a specific enzyme

More Recent History of Gene Expression Many proteins are composed of several polypeptides, each of which has its own gene Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis

The Structure of RNA single stranded working copy of one gene Disposable RNA is the intermediate between genes and the proteins for which they code

The Structure of RNA one gene from DNA can produce many strands of RNA instead of thymine (T), uracil (U) RNA has A, U, C, G

Transcription and Translation Transcription is the synthesis of RNA under the direction of DNA nucleus Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA cytoplasm Ribosomes are the sites of translation

Transcription and Translation Overview In prokaryotes, mRNA produced by transcription is immediately translated without more processing In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA

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

DNA TRANSCRIPTION mRNA (a) Bacterial cell Fig. 17-3a-1 Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information (a) Bacterial cell

DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Fig. 17-3a-2 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information (a) Bacterial cell

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

17.2 Transcription DNA  RNA

Types of RNA three types messenger RNA (mRNA) ribosomal RNA (rRNA) transfer RNA (tRNA)

Transcription requires enzyme RNA polymerase RNA polymerase binds to DNA and separates the strands Similar to DNA replication

Transcription RNA is made in the 5’ – 3’ direction RNA polymerase uses one strand of DNA as a template to make RNA RNA is made in the 5’ – 3’ direction How is the DNA read?

Transcription The DNA sequence where RNA polymerase attaches is called the promoter In bacteria, the sequence signaling the end of transcription is called the terminator The stretch of DNA that is transcribed is called a transcription unit

Completed RNA transcript Fig. 17-7 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation Elongation Nontemplate strand of DNA RNA nucleotides 5 3 RNA polymerase 3 5 RNA transcript Template strand of DNA Unwound DNA 3 2 Elongation 3 end Rewound DNA 5 5 3 3 3 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 5 Direction of transcription (“downstream”) RNA transcript Template strand of DNA 3 Termination Newly made RNA 5 3 3 5 5 3 Completed RNA transcript

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

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

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

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

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

Transcription Overview http://www.youtube.com/watch?v=WsofH466lqk

Three Stages of Transcription Initiation Elongation Termination

Initiation Signaled by promoters Transcription factors mediate the binding of RNA polymerase and the initiation of transcription Transcription initiation complex: all transcription factors and RNA polymerase II bound to a promoter A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

Several transcription factors must bind to the DNA before RNA Fig. 17-8 1 A eukaryotic promoter includes a TATA box Promoter Template 5 3 3 5 TATA box Start point Template DNA strand 2 Several transcription factors must bind to the DNA before RNA polymerase II can do so. Transcription factors 5 3 3 5 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. Figure 17.8 The initiation of transcription at a eukaryotic promoter RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex

Elongation RNA polymerase untwists the DNA, 10 to 20 bases at a time Transcription progresses at a rate of 40 nucleotides per second in eukaryotes A gene can be transcribed simultaneously by several RNA polymerases

Termination Bacteria: the polymerase stops transcription at the end of the terminator Eukaryotes: the polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA chain Pre-mRNA will be cleaved when the RNA polymerase II transcribes a polyadenylation sequence (AAUAAA on the pre-mRNA) the polymerase eventually falls off the DNA

17.3 RNA Processing

RNA Processing Enzymes in the eukaryotic nucleus modify pre-mRNA During RNA processing, both ends of the primary transcript are usually altered Some interior parts of the molecule are cut out, and the other parts spliced together

RNA Modifications: the ends Each end of a pre-mRNA molecule is modified in a particular way: The 5’ end receives a modified guanine nucleotide 5’ cap The 3’ end gets a poly-A tail (made of 50-250 more adenine nucleotides)

RNA Modifications: the ends These modifications share several functions: Facilitate the export of mRNA Protection of the mRNA from hydrolytic enzymes They help ribosomes attach to the 5’ end

Protein-coding segment Polyadenylation signal 5 3 Fig. 17-9 Protein-coding segment Polyadenylation 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

RNA Splicing Noncoding stretches of nucleotides that lie between coding regions intervening sequences or introns Exons: eventually expressed, translated into amino acid sequences RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

RNA Processing RNA will copy both introns and exons from the DNA Introns need to be cut out to make a working piece of RNA Remaining pieces of exons will be spliced together to form the final mRNA sequence

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 Figure 17.10 RNA processing: RNA splicing 1 146 5 UTR 3 UTR

In some cases, RNA splicing is carried out by spliceosomes Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Fig. 17-11-1 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Figure 17.11 The roles of snRNPs and spliceosomes in pre-mRNA splicing

RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Fig. 17-11-2 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Figure 17.11 The roles of snRNPs and spliceosomes in pre-mRNA splicing

RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Fig. 17-11-3 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Figure 17.11 The roles of snRNPs and spliceosomes in pre-mRNA splicing Spliceosome components Cut-out intron mRNA 5 Exon 1 Exon 2

Ribozymes Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

RNA can function as an enzyme It can form a three-dimensional structure because of its ability to base pair with itself Some bases in RNA contain functional groups RNA may hydrogen-bond with other nucleic acid molecules

Alternative RNA Splicing Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing Number of different proteins an organism can produce is much greater than its number of genes

Protein Domains Proteins often have a modular architecture consisting of discrete regions called domains In many cases, different exons code for the different domains in a protein Exon shuffling may result in the evolution of new proteins Protein will fold differently = act differently

Reading the Genetic Code

The Genetic Code Genetic code, or language of mRNA instructions, is read three letters at a time

The Genetic Code Each three letter sequence, or “word”, codes for one amino acid these three letter words are known as codons Codons are found on the mRNA The protein is determined by the order of each amino acid in the sequence

The Genetic Code There are 64 possible codons One codon, AUG, is known as the “start” codon for protein synthesis There are also three possible sequences that are called “stop” codons Show where polypeptide ends

Translation

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

Translation Making a protein from a strand of mRNA

tRNA Consists of a single RNA strand that is only about 80 nucleotides long Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf

Figure 17.14 The structure of transfer RNA (tRNA) 3 Amino acid attachment site 5 Hydrogen bonds Anticodon (a) Two-dimensional structure 5 Amino acid attachment site 3 Figure 17.14 The structure of transfer RNA (tRNA) Hydrogen bonds 3 5 Anticodon Anticodon (c) Symbol used in this book (b) Three-dimensional structure

In order to have accurate translation… First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase Second: a correct match between the tRNA anticodon and an mRNA codon Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon

Aminoacyl-tRNA Amino acid synthetase (enzyme) Fig. 17-15-1 P P P Adenosine ATP Figure 17.15 An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA

Aminoacyl-tRNA Amino acid synthetase (enzyme) Fig. 17-15-2 P P P Adenosine ATP P Adenosine P P i P i P i Figure 17.15 An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA

Aminoacyl-tRNA Amino acid synthetase (enzyme) tRNA Aminoacyl-tRNA Fig. 17-15-3 Aminoacyl-tRNA synthetase (enzyme) Amino acid P P P Adenosine ATP P Adenosine tRNA P P i Aminoacyl-tRNA synthetase P i P i tRNA Figure 17.15 An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA P Adenosine AMP Computer model

Aminoacyl-tRNA Amino acid synthetase (enzyme) tRNA Aminoacyl-tRNA Fig. 17-15-4 Aminoacyl-tRNA synthetase (enzyme) Amino acid P P P Adenosine ATP P Adenosine tRNA P P i Aminoacyl-tRNA synthetase P i P i tRNA Figure 17.15 An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA P Adenosine AMP Computer model Aminoacyl-tRNA (“charged tRNA”)

Ribosomes Ribosomal RNA (rRNA) Large subunit Small subunit

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

(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

Ribosomes A ribosome has three binding sites for tRNA: The P site (peptidyl-tRNA binding site) holds the tRNA that carries the growing polypeptide chain The A site (Aminoacyl-tRNA binding site) holds the tRNA that carries the next amino acid to be added to the chain The E site (exit site) is where discharged tRNAs leave the ribosome

Three Parts of Translation Initiation Elongation Termination

Steps of Translation mRNA attaches to a ribosome a small ribosomal subunit binds with mRNA and a special initiator tRNA small subunit moves along the mRNA until it reaches the start codon (AUG) initiation factors bring in the large subunit that completes the translation initiation complex

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 Figure 17.17 The initiation of translation Start codon Small ribosomal subunit mRNA binding site Translation initiation complex

Elongation Amino acids are added one by one to the preceding amino acid Each addition involves proteins called elongation factors and occurs in three steps: codon recognition peptide bond formation Translocation

Elongation: Codon Recognition 2. tRNA brings the correct amino acid to the ribosome each tRNA carries only one type of amino acid the anticodon sequence is complimentary to the codon

Elongation: Peptide Bond Formation 3. Ribosome forms a peptide bond between the amino acids (forming a protein)

Elongation: Translocation 4. The tRNA from the first amino acid is then released from the ribosome via the E site 5. Ribosome then moves to the next codon and a new tRNA enters the A site

Translation Continues! 6. Polypeptide chain grows until the ribosome reaches a “stop” codon Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome

Translation Complete! 7. The A site accepts a protein called a release factor The release factor causes the addition of a water molecule instead of an amino acid Releases the polypeptide, and the translation assembly then comes apart

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

Polypeptide chains are modified after translation Completed proteins are targeted to specific sites in the cell

During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape Proteins may also require post-translational modifications before doing their job Some polypeptides are activated by enzymes that cleave them Other polypeptides come together to form the subunits of a protein

Ribosomes Free ribosomes: in cytosol Bound ribosomes: on surface of ER Make proteins to be excreted from the cell All transcription occurs in the cytoplasm Polypeptides destined for the ER or for secretion are marked by a signal peptide A signal-recognition particle (SRP) binds to the signal peptide The SRP brings the signal peptide and its ribosome to the ER

Mutations Mutations are changes in the genetic material of a cell or virus Point mutations are chemical changes in just one base pair of a gene Substitution Insertion or Deletion

Effects of Substitutions 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 necessarily the right amino acid Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

Effects of Insertions and Deletions More harmful than substitutions Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation

Mutagens Spontaneous mutations can occur during DNA replication, recombination, or repair Mutagens are physical or chemical agents that can cause mutations

Gene Expression in Archae, Bacteria, Eukaryotes Use your text book to compare and contrast the cellular processes of gene expression in archaea, bacteria and eukarya

Genes and Proteins genes code for proteins which are what carry out expression of these genes proteins code for enzymes which cause certain reactions to take place these reactions are what cause traits!

Protein Structure

Protein Structure Primary (1°)structure: amino acid sequence from translation Secondary (2°) structure: alpha (α) helix and beta (β) sheets

Protein Structure Tertiary (3º) structure: three dimensional structure Each protein has its own unique tertiary structure

Protein Structure Quaternary (4°) structure: multiple tertiary structures form a complex The protein will change confirmation (shape) if not being used