RNA & Gene Expression

1. RNA & Gene Expression • Gene: A segment of DNA that specifies the amino acid sequence of a polypeptide • DNA does not directly control protein synthesis, instead its information is transcribed into RNA • The “Central Dogma”:

2. RNA properties • RNA (ribonucleic acid)

3. Types of RNA • Three Classes of RNA • Messenger RNA (mRNA) • Takes a message from DNA to the ribosomes • strand • Ribosomal RNA (rRNA) • Makes up ribosomes (along with proteins) • globular • Transfer RNA (tRNA) • Transfers amino acids to ribosomes • Hairpin shape

4. Gene Expression • Gene Expression Requires Two Steps: • Transcription • Is the synthesis of RNA under the direction of DNA • Produces messenger RNA (mRNA) • Translation • Is the actual synthesis of a polypeptide, which occurs under the direction of mRNA • Occurs on ribosomes http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation1.html

5. Ribonucleic Acid • Why would the cell want to have an intermediate between DNA and the proteins it encodes? • The DNA can then stay pristine and protected, away from the caustic chemistry of the cytoplasm. • Gene information can be amplified by having many copies of an RNA made from one copy of DNA. • Regulation of gene expression can be effected by having specific controls at each element of the pathway between DNA and proteins. • The more elements there are in the pathway, the more opportunities there are to control it in different circumstances.

6. 24.2 Gene Expression • Transcription • During transcription, a segment of the DNA serves as a template for the production of an RNA molecule • Messenger RNA (mRNA) • RNA polymerase (enzyme) binds to a promoter (“start” sequence) • DNA helix is opened so complementary base pairing can occur • RNA polymerase joins new RNA nucleotides in a sequence complementary to that on the DNA, in a 5’ to 3’ direction

7. Transcription of DNA to form mRNA

8. Messenger RNA • mRNA - of the 64 possible 3-base combinations: • 61 code for the twenty different amino acids • 3 code for "stop"; i.e. chain termination • Specific nucleotide sequences call for “start” of transcription (usually  AUG = methionine) = PROMOTOR sequence • “stop” of mRNA synthesis = TERMINATION sequence (UAA, UGA, UAG) • Finished mRNA strands are ~500-10,000 nucleotides long

9. Gene 2 DNA molecule Gene 1 Gene 3 DNA strand (template) 5 3 A C C T A A A C C G A G TRANSCRIPTION A U C G C U G G G U U U 5 mRNA 3 Codon TRANSLATION Gly Phe Protein Trp Ser Figure 17.4 Amino acid • During transcription • The gene determines the sequence of bases along the length of an mRNA molecule

10. 24.2 Processing of mRNA • After Transcription • Primary “Pre-”mRNA must be modified into mature mRNA • Introns are intragene segments (often, junk) • Exons are the portion of a gene that is expressed • Intron sequences are removed, and a poly-A tail is added • Ribozyme splices exon segments together • http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation20.html

11. mRNA Processing pre-RNA must be modified before translation

12. The Functional and Evolutionary Importance of Introns • The presence of introns • Allows for alternative RNA splicing • Animations of RNA processing: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/bio25.swf::Processing%20of%20Gene%20Information%20-%20Prokaryotes%20versus%20Eukaryotes • http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/bio30.swf::How%20Spliceosomes%20Process%20RNA

13. Transposons • Transposons • “jumping genes” • Can move to new locations and disrupt gene sequences

14. 24.2 Gene Expression • Translation • The Genetic Code • Triplet code: each 3-nucleotide unit of a mRNA molecule is called a codon • There are 64 different mRNA codons • 61 code for particular amino acids • Redundant code; some amino acids have numerous code words • Provides some protection against mutations • 3 are stop codons signal polypeptide termination

15. Messenger RNA Codons

16. Overview of Gene Expression Protein synthesis http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis

17. 24.2 Gene Expression • Transfer RNA • tRNA transports amino acids to the ribosomes (creates polypeptide chain) • Single stranded nucleic acid that correlates a specific nucleotide sequence with a specific amino acid • Amino acid binds to one end, the opposite end has an anticodon • the order of mRNA codons determines the order in which tRNA brings in amino acids Protein synthesis http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis

18. Transfer RNA: Amino Acid Carrier

19. rRNA • Ribosomal RNA is the most abundant type of RNA in cells • Ribosomes: comprised of subunits 2/3 RNA, 1/3 protein • Two populations of ribosomes are evident in cells, Free and bound

20. “Bound” vs “Free” Ribosomes • Free ribosomes are located in the cytoplasm of the cell. They are not attached to any structure, but they may group together with other ribosomes to form polysomes (polyribosomes). In the cytoplasm, ribosomes are free floating. They can move all around the cell. • Bound ribosomes are located on the surface of the endoplasmic reticulum. The endoplasmic reticulum that contains ribosomes is described as the rough endoplasmic reticulum because of the bumpy surface. Bound ribosomes can not move to other areas of the cell. They are attached to the cytosolic side of the endoplasmic reticulum. • Free ribosomes produce proteins for the cell, while bound ribosomes produce proteins that are transported out of the cell.

21. Completed polypeptide Growing polypeptides Incoming ribosomal subunits Start of mRNA (5 end) Polyribosome End of mRNA (3 end) (a) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mRNA 0.1 µm (b) This micrograph shows a large polyribosome in a prokaryotic cell (TEM). Figure 17.20a, b Polyribosomes • A number of ribosomes can translate a single mRNA molecule simultaneously • Forming a polyribosome

22. P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams. • The ribosome has three binding sites for tRNA • The P site • The A site • The E site E P A Figure 17.16b

23. Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look Quicktime movie: http://carbon.cudenver.edu/~bstith/transla.MOV Narrated animation: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis Interactive practice: http://learn.genetics.utah.edu/content/begin/dna/transcribe/

24. 24.2 Gene Expression • Ribosome and Ribosomal RNA • Ribosome has a binding site for mRNA and for 2 tRNAs • Facilitates complementary base pairing • Ribosome moves along mRNA and new tRNAs come in and line up in order • This brings amino acids in line in a specific order to form a polypeptide • Several ribosomes may move along the same mRNA • Multiple copies of a polypeptide may be made • The entire complex is called a polyribosome

25. Growing polypeptide Amino end Next amino acid to be added to polypeptide chain tRNA 3 mRNA Codons 5 (c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. Translation (Building a polypeptide) requires Three Steps: • Initiation (requires energy) • Elongation (requires energy) • Termination

26. Summary of Gene Expression

27. 24.2 Gene Expression • Genes and Gene Mutations • A gene mutation is a change in the sequence of bases within a gene. • Gene mutations can lead to malfunctioning proteins in cells.

28. 24.2 Gene Expression • Genes and Gene Mutations • Causes of Mutations • Errors in replication • Rare • DNA polymerase “proofreads” new strands and errors are cleaved out • Mutagens • Environmental influences • Radiation, UV light, chemicals • Rate is still fairly low because DNA repair enzymes monitor and repair DNA

29. Types of Gene Mutations Point Mutations • The substitution of one nucleotide for another • Missense mutations • a point mutation in which a single nucleotide is changed, resulting in a codon that codes for a different amino acid • Missense mutations are responsible for about 75% of the mutations in the p53 gene. Mutations of this gene are responsible for about 30-50% of cancers in humans • Silent mutations • Nonsense mutations • mutations that change an amino acid to a stop codon

30. Types of Gene Mutations • Frameshift Mutations • One or more nucleotides are inserted or deleted • Results in a polypeptide that codes for the wrong sequence of amino acids • Codons must be read in the correct reading frame for the specified polypeptide to be produced