1. Gene Activity 1 Ch. 12-b Outline – Gene Activity

2. Gene Activity 2 Function of Genes Archibald Garrod: ● First to propose a link between genes and proteins and metabolic diseases. ● Inherited disorders could be caused by lack of a particular enzyme in a metabolic pathway.

3. Gene Activity 3 Genes Specify Enzymes   Beadle and Tatum: ­ ­Experiments on fungus Neurospora crassa ­ ­Induced mutations with X-rays. Spores were no longer able to grow on minimal media. ­ ­Figured out which enzymes were lacking & found that each mutant strain only had 1 defective gene ­ ­Proposed that each gene specifies the synthesis of one enzyme ­ ­One-gene-one-enzyme hypothesis

4. 4 Beadle & Tatum Experiment

5. Gene Activity 5 Genes Specify a Polypeptide   A gene is a segment of DNA that specifies the sequence of amino acids in a polypeptide   Suggests that genetic mutations cause changes in the primary structure of a protein   Examples of genetic mutations that cause disease: 1. Sickle cell disease : a change in DNA causes hemoglobin to have one different amino acid which changes the function of the entire protein. (This was discovered by Linus Pauling and Harvey Itano in 1949)

6. 6 Sickle-Cell Disease in Humans

7. Gene Activity 7 Protein Synthesis: From DNA to RNA to Protein The mechanism of gene expression   DNA in genes specify information, but information is not structure and function   Genetic info is expressed into structure & function through protein synthesis The expression of genetic info into structure & function:   DNA in gene controls the sequence of nucleotides in an RNA molecule   RNA controls the primary structure of a protein

8. Gene Activity 8 Types of RNA (see transparency here) RNA is a polymer of RNA nucleotides RNA nucleotides are of four types: Uracil, Adenine, Cytosine, and Guanine Uracil (U) replaces thymine (T) of DNA Types of RNA ­ ­Messenger (mRNA) - Takes genetic message from DNA in nucleus to ribosomes in cytoplasm ­ ­Ribosomal (rRNA) - Makes up ribosomes which read the message in mRNA ­ ­Transfer (tRNA) - Transfers appropriate amino acid to ribosome when “instructed”

9. 9 Structure of RNA

10. 10 Three Types of RNA

11. Gene Activity 11 Steps in Gene Expression: There are two steps in gene expression: 1. 1.Transcription ● DNA serves as a template for RNA formation 2. 2.Translation ● mRNA transcript directs the creation of a sequence of amino acids in a polypeptide

12. 12 Overview of Gene Expression

13. Gene Activity 13 The Genetic Code Each of the 20 amino acids found in proteins is specified by one or more codons   Genetic alphabet has only four “letters”: U,A,C,G   Codons in the genetic code are all three bases long ­ ­There are 64 possible arrangements of four symbols taken three at a time   Often referred to as triplets ­ ­Genetic language has 64 “words” that code for 20 amino acids

14. Gene Activity 14 Finding the Genetic Code Nirenberg & Matthei (1961) 1. 1.They found that cell enzymes could be used to construct synthetic RNA. 2. 2.Then found that these RNA could be translated in a test tube into polypeptides 3. 3.The first synthetic RNA was made of only U. ● This resulted in a polypeptide made up only of phenylalanine. 4. They repeated this with all possible triplet combinations until they constructed the genetic code table.

15. 15 The Genetic Code (mRNA) CAG

16. Gene Activity 16 Genetic Code is Universal   All organisms use the same genetic code - Slight differences in mitochondria and chloroplasts DNA, however.   Degenerate (redundant) ­ ­There are 64 codons available for 20 amino acids ­ ­Most amino acids encoded by two or more codons   Contains start and stop signals   Makes it possible to transfer genes between different organisms.

17. Gene Activity 17 Steps in Gene Expression: First = Transcription All types of RNA are created by transcription   DNA unzips and exposes unpaired bases   One side serves as template for mRNA formation. This strand is also called the sense strand.   Loose RNA nucleotides bind to template DNA bases using the C=G & A=U rule This occurs with the help of RNA polymerase. ● The strand of DNA that is not transcribed is called the noncoding strand or nonsense strand.

18. Gene Activity 18 Steps in Gene Expression: Transcription (cont’d) Transcription begins when RNA polymerase attaches to a region of DNA known as the promoter.   RNA polymerase joins the nucleotides together in the 5’ to 3’ direction. - It only adds a nucleotide to the 3’ end of the RNA being formed.   Elongation of mRNA continues until RNA polymerase comes to a DNA stop sequence.

19. Gene Activity 19 Steps in Gene Expression: Transcription (cont’d)   When entire gene is transcribed into mRNA, result is an mRNA transcript of the gene   The base sequence in the mRNA is complementary to the base sequence in DNA   Many RNA polymerase molecules can be working at the same time. Thus, a cell can produce thousands of copies of the same mRNA within a short period of time.

20. 20 Transcription

21. 21 RNA Polymerase

22. Gene Activity 22 Processing Messenger RNA In eukaryotes the primary mRNA transcript is modified before it leaves the nucleus   RNA splicing occurs: ­ ­Primary transcript consists of:   Some segments that will not be expressed (introns)   Segments that will be expressed (exons) ­ ­Performed by spliceosome complexes in nucleoplasm   Introns are excised (cut out)   Remaining exons are spliced back together

23. Gene Activity 23 Processing Messenger RNA (cont’d)   Modifications to ends of primary transcript: ­ ­Cap of modified guanine on 5′ end ­ ­Poly-A tail of 150+ adenines on 3′ end Result is mature mRNA transcript

24. 24 mRNA Processing in Eukaryotes

25. Gene Activity 25 Functions of Introns Eukaryotes have many introns while prokaryotes don’t.   In humans up to 95% of genes are introns. ­ ­Genome has only about 25,000 coding genes Possible functions of introns: ­ ­Exons might combine in various combinations ­ ­Would allow different mRNAs to result from one segment of DNA   Introns might regulate gene expression

26. Gene Activity 26 Steps in Gene Expression: Second = Translation ● Takes place in cytoplasm. ● Codons in mRNA direct the sequence of amino acids in a polypeptide. One “language” is translated into another. ● Involves all three types of RNA ● tRNA - Single-stranded RNA - Transfers amino acids to the ribosomes. - There is at least one tRNA for each of the 20 amino acids found in proteins.

27. Gene Activity 27 Steps in Gene Expression: Role of tRNA tRNA molecules have two binding sites:   An amino acid binds to the 3’ end of the tRNA - This is helped along by an enzyme called aminoacyl-tRNA synthetase.   The opposite end of the tRNA contains an anticodon: - This is a group of 3 bases that are complementary to a specific codon of mRNA All tRNA molecules with a specific anticodon will always bind with the same amino acid

28. 28 Structure of tRNA

29. Gene Activity 29 Role of Ribosomes Ribosomal RNA (rRNA):   Produced from a DNA template in the nucleolus   Combined with proteins into large and small ribosomal subunits A completed ribosome has three binding sites to facilitate pairing between tRNA and mRNA   The E (for exit) site   The P (for peptide) site, and   The A (for amino acid) site

30. 30 Ribosomal Binding Sites

31. 31 Ribosomal Structure and Function

32. Gene Activity 32 Steps in Gene Expression: Second = Translation   An mRNA transcript migrates to rough endoplasmic reticulum   Associates with the rRNA of a ribosome   The ribosome “reads” the information in the transcript   Ribosome directs various species of tRNA to bring in their specific amino acid “fares”   tRNA specified is determined by the code being translated in the codons of mRNA transcript

33. Gene Activity 33 Steps in Translation: #1 - Initiation Components necessary for initiation are:   Small & large ribosomal subunits   mRNA transcript   Initiator tRNA   Initiation factors (special proteins that bring the above together) Initiator tRNA:   Always has the UAC anticodon   Always carries the amino acid methionine   Capable of binding to the P site

34. Gene Activity 34 Steps in Translation: #1 - Initiation ● Small ribosomal subunit attaches to mRNA transcript ● Initiator tRNA (UAC) attaches to P site - Beginning of transcript always has the START codon (AUG). So first tRNA always carries the amino acid methione. ● Large ribosomal subunit joins the small subunit

35. 35 Steps in Translation: #1 - Initiation

36. Gene Activity 36 Steps in Translation: #2 - Elongation “Elongation” refers to the growth in length of the polypeptide ● tRNA molecules bring their amino acid fares to the ribosome ­ ­ They must have the anticodon complementary to the mRNA codon being read ­ ­Joins the ribosome at it’s A site   Methionine of initiator is connected to amino acid of 2 nd tRNA by peptide bond

37. Gene Activity 37 Steps in Translation: #2 – Elongation (cont’d) Second tRNA moves over to the P site (translocation) Spent initiator moves to E site and exits Ribosome reads the next codon in the mRNA ­ ­Joins the ribosome at it’s A site   Dipeptide on 2 nd amino acid is connected to amino acid of 3 rd tRNA by peptide bond - A ribozyme and energy is needed to bring about this transfer.

38. 38 Steps in Translation: # 2 Elongation

39. Gene Activity 39 Steps in Translation: #3 – Termination Final step in protein synthesis: ● Ribosome reads the STOP codon at the end of the mRNA. These are the following codons:   UAA, UAG, or UGA   These do not code for an amino acid Polypeptide is released from last tRNA by release factor Ribosome releases mRNA and dissociates into subunits mRNA read by another ribosome

40. 40 Steps in Translation: #3 - Termination

41. 41 Summary of Gene Expression (Eukaryotes)

42. 42 DNA Replication Video I http://www.courses.fas.harvard.edu/~biotext/ani mations/replication1.html DNA Replication Video II http://highered.mcgraw-hill.com/olc/dl/120076/bio23.swf Protein Synthesis Video http://highered.mcgraw- hill.com/olc/dl/120077/micro06.swf