Biol115 The Thread of Life

Transcription

1 Biol115 The Thread of Life Lecture 6 Getting the message: DNA transcription Understanding how DNA transmits all it knows about cancer, physics, dreaming and love will keep man searching for some time ~David R. Brower

2 Principles of Biology Chapter Gene Expression (The expression of genes) Chapter Transcription Biol115_2014_Lecture 6 2

3 Objectives Explain the processes that occur during the three phases of transcription. Describe the molecular factors that aid in transcription. Relate the importance of specific sequences on the DNA molecule to the process of transcription. Describe the differences between eukaryotic and prokaryotic transcription. Describe RNA processing. Key terms: elongation, initiation, intron, promoter, RNA polymerase, TATA box, termination, transcription, transcription initiation complex, transcription start site Biol115_2014_Lecture 6 3

4 Gene expression: the cookbook analogy kbook-clipart.gif ipes/i/recipes/sl/02/11/ch ocolate-cake-sl l.jpg DNA transcription mrna translation polypeptide/protein Biol115_2014_Lecture 6 4

5 Basic principles of transcription Transcription is the synthesis of RNA under the direction of DNA Transcription is mediated by the enzyme, RNA polymerase Transcription produces messenger RNA (mrna) RNA polymerase and transcription Biol115_2014_Lecture 6 5

6 Basic principles of transcription 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 Biol115_2014_Lecture 6 6

7 Synthesis of an RNA transcript The three stages of transcription: Initiation Elongation Termination Biol115_2014_Lecture 6 7

8 RNA polymerase binding and initiation of transcription Promoters signal the initiation of RNA synthesis Transcription factors mediate the binding of RNA polymerase and the initiation of transcription The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes Biol115_2014_Lecture 6 8

9 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 simultaneously by several RNA polymerases Biol115_2014_Lecture 6 9

10 Termination of transcription The mechanisms of termination are different in bacteria and eukaryotes In bacteria, the polymerase stops transcription at the end of the terminator In eukaryotes, the polymerase continues transcription after the pre-mrna is cleaved from the growing RNA chain; the polymerase eventually falls off the DNA Biol115_2014_Lecture 6 10

11 Transcription in prokaryotes Multiple RNA polymerases transcribing the same gene simultaneously! Nature 417, Biol115_2014_Lecture 6 11

12 RNA polymerase binding and initiation of transcription In eukaryotes, transcription factors mediate the binding of RNA polymerase and the initiation of transcription A sequence in the upstream regulatory region, called a TATA box, is crucial in forming the initiation complex in eukaryotes Biol115_2014_Lecture 6 12

13 The eukaryotic promoter Promoters are the start sites of transcription. Eukaryotic promoters can include the TFIIB recognition element, the TATA box, the initiator element and the downstream promoter element. Not all of these are present in every promoter. Biol115_2014_Lecture 6 13

14 Termination of transcription RNA polymerase stops transcribing at the end of a gene stop defined by termination signals (example shown) Termination signals (Gould and Keeton, Biological Sciences) Hairpin loops and poly-u tails (Gould and Keeton, Biological Sciences) Biol115_2014_Lecture 6 14

15 Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre-mrna before the genetic messages are dispatched to the cytoplasm 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 Biol115_2014_Lecture 6 15

16 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 These modifications share several functions: They seem to facilitate the export of mrna They protect mrna from hydrolytic enzymes They help ribosomes attach to the 5 end (for translation) Biol115_2014_Lecture 6 16

17 Insert Fig P336 Biol115_2014_Lecture 6 17

18 Split genes and RNA splicing Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions These noncoding regions are called intervening sequences, or introns The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences RNA splicing removes introns and joins exons, creating an mrna molecule with a continuous coding sequence Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snrnps) that recognise the splice sites Biol115_2014_Lecture 6 18

19 Example of mrna splicing E = exon I = intron DNAASDFASISASDFTHECGHDTHREADARETVTOFSRVTLIFE pre-mrna E1 I1 E2 I2 E3 I3 E4 I4 E5 I5 E6 mature mrna DNAISTHETHREADOFLIFE Biol115_2014_Lecture 6 19

20 Insert Fig P337 Biol115_2014_Lecture 6 20

21 RNA splicing In eukaryotes, coding regions, or exons, are interspersed with non-coding introns. Introns are removed, and exons are joined together during RNA splicing to produce a mature mrna molecule. Biol115_2014_Lecture 6 21

22 mrna for the egg protein, ovalbumin, being spliced to remove introns Gene Pre-mRNA mrna Factor 8 200,000 nt 10,000 nt Duchenne muscular dystrophy 2,000,000 nt 16,000 nt Biol115_2014_Lecture 6 22

23 The functional and evolutionary importance of introns 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 Biol115_2014_Lecture 6 23

24 The functional and evolutionary importance of introns Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing Such variations are called alternative RNA splicing Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes Biol115_2014_Lecture 6 24

25 Alternative splicing: expressing several proteins from a single gene. Even though it is translated from a single gene, the information within an mrna transcript can be rearranged during mrna splicing. The final mrna would be translated into a protein with a different number or order of protein subunits than what was coded for by the pre-spliced mrna. The Drosophila Dscam gene, involved in adhesion between neurons, contains 4 clusters of exons, each with array of possible exons. These are spliced into the mrna in an exclusive fashion, so that only one of each of the possible exons is represented. If all combinations of these exons are used in alternative splicing, the Dscam gene can produce 38,016 different proteins. Biol115_2014_Lecture 6 25

26 You should now be able to: 1. Briefly explain how information flows from gene to protein. 2. Compare transcription in bacteria and eukaryotes. 3. Include the following terms in a description of transcription: mrna, RNA polymerase, the promoter, the terminator, the transcription unit, initiation, elongation, termination, splicing and introns. 4. Explain three types of post-transcriptional processing of eukaryotic pre-mrna. 5. Suggest reasons for the occurrence of introns in eukaryotic genes and the possible evolutionary benefits that they confer. Biol115_2014_Lecture 6 26