1. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Gene expression is the process by which DNA directs protein synthesis, includes two stages: – Transcription – Translation

2. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Basic Principles of Transcription and Translation Transcription is the synthesis of RNA under the direction of DNA Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA Ribosomes are the sites of translation TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Prokaryotic cell

3. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings In an eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA before leaving the nucleus The finished mRNA instructs the ribosomes in the cytoplasm of the cell to make proteins Cells are governed by a cellular chain of command: DNA  mRNA  protein DNA  RNA  protein

4. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide DNA Pre-mRNA Prokaryotic cell Nuclear envelope mRNA TRANSLATION TRANSCRIPTION RNA PROCESSING Ribosome Polypeptide Eukaryotic cell

5. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings During transcription, a DNA strand called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript (mRNA) During translation, the mRNA base triplets, called codons, are read in the 5 to 3 direction Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide

6. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings DNA molecule Gene 1 Gene 2 Gene 3 DNA strand (template) 3 TRANSCRIPTION Codon mRNA TRANSLATION Protein Amino acid 3 5 5

7. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Second mRNA base First mRNA base (5 end) Third mR NA base (3 end)

8. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Transcription is the DNA-directed synthesis of RNA: Molecular Components of Transcription RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart, adds and binds the RNA nucleotides RNA synthesis follows the same base- pairing rules as DNA, except uracil substitutes for thymine

9. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The three stages of transcription: – Initiation – Elongation – Termination The DNA sequence where RNA polymerase attaches is called the promoter. The stretch of DNA that is transcribed is called a transcription unit. Transcription

10. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Promoter 3 5 Transcription unit DNA Initiation RNA polymerase Start point Template strand of DNA RNA tran- script Unwound DNA Elongation 3 3 5 3 5 5 35 Rewound DNA 5 3 3 5 3 5 RNA transcript Termination 35 5 3 Completed RNA transcript Elongation Non-template strand of DNA RNA polymerase RNA nucleotides 3 end 3 5 5 Newly made RNA Template strand of DNA Direction of transcription (“downstream”)

11. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Eukaryotic cells modify mRNA after transcription The outcome of the transcription process in eukaryotic cells is the pre-mRNA Enzymes in the eukaryotic nucleus modify pre-mRNA before it is dispatched to the cytoplasm, through the pores of the nucleus, as mRNA

12. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Translation is the RNA-directed synthesis of a polypeptide: Molecular Components of Translation 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 Amino acid attachment site Hydrogen bonds 3 5 Two-dimensional structure Anticodon

13. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Amino acid attachment site Hydrogen bonds 3 5 Two-dimensional structure Anticodon Amino acid attachment site 3 5 Hydrogen bonds Anticodon Symbol used in this book Three-dimensional structure 35 tRNA

14. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Ribosomes Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA)

15. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings A ribosome has three binding sites for tRNA: – The P site holds the tRNA that carries the growing polypeptide chain – The A site holds the tRNA that carries the next amino acid to be added to the chain – The E site is the exit site, where discharged tRNAs leave the ribosome Ribosomes

16. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings P site (Peptidyl-tRNA binding site) E site (Exit site) mRNA binding site A site (Aminoacyl- tRNA binding site) Large subunit Small subunit Schematic model showing binding sites EPA Ribosomes

17. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 17-16c Amino end mRNA 5 3 Growing polypeptide Next amino acid to be added to polypeptide chain tRNA Codons Schematic model with mRNA and tRNA E

18. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Translation (Building a Polypeptide) The three stages of translation: – Initiation – Elongation – Termination All three stages require protein “factors” that aid in the translation process

19. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Ribosome Association and Initiation of Translation The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits Met GTP Initiator tRNA mRNA 5 3 mRNA binding site Small ribosomal subunit Start codon P site 5 3 Translation initiation complex E A Large ribosomal subunit GDP Met

20. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Ribosome Association and 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 so the initiator tRNA occupies the P site

21. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Met GTP Initiator tRNA mRNA 5 3 mRNA binding site Small ribosomal subunit Start codon P site 5 3 Translation initiation complex E A Large ribosomal subunit GDP Met Initiation of Translation

22. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Ribosome ready for next aminoacyl tRNA mRNA 5 Amino end of polypeptide E P site A site 3 2 2 GDP E PA GTP GDP E PA E PA Polypeptide Elongation

23. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 causes the addition of a water molecule instead of an amino acid This reaction releases the polypeptide, and the translation assembly then comes apart

24. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 3 The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. The two ribosomal subunits and the other components of the assembly dissociate. Release factor Stop codon (UAG, UAA, or UGA) 5 3 5 3 5 Free polypeptide When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. Termination of Translation http://www.youtube.com/watch?v=D3fOXt4MrOM From DNA to Protein

25. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Polysomes Several ribosomes can translate an mRNA at the same time, forming what is called a polysome. Polysomes make it possible to produce many polypeptides simultaneously from a single mRNA.

26. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 17.7: Point mutations can affect protein structure and function 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 The change of a single nucleotide in a DNA template strand leads to production of an abnormal protein

27. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Wild-type hemoglobin DNA mRNA 3553 5335 Mutant hemoglobin DNA mRNA Normal hemoglobinSickle-cell hemoglobin