Transcription and Translation

1. Transcription and Translation Chapter 16

2. Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Polypeptide Information Flow in the Cell

3. 3 1 2 Promoter Transcription unit Start point 5 3 3 5 RNA polymerase Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. DNA Template strand of DNA Unwound DNA RNA transcript 5 3 3 5 Elongation. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5 3 . In the wake of transcription, the DNA strands re-form a double helix. Rewound RNA 5 3 3 5 RNA transcript 3 5 Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA. 5 3 3 5 3 5 Completed RNA transcript Transcription • The stages of transcription are • Initiation • Elongation • Termination

4. Transcription in Bacteria

5. Transcription • First step in converting genetic information into proteins. • Copying DNA to make an RNA messenger • DNA-directed synthesis of RNA • Catalyzed by RNA polymerase • Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine

6. Transcription

7. RNA Polymerase • Pries the DNA strands apart and hooks together the RNA nucleotides • Synthesizes the RNA strand in the 5'  3' direction • RNA strand is complementary to the DNA template strand. • Provides single-stranded RNA copy of the DNA • This strand is the template strand, the other the non-template strand.

8. Bacterial RNA Polymerase • Globular enzyme with several channels and a magnesium atom in its active site • Holoenzyme made up of the core enzyme, which has the ability to synthesize RNA • And a regulatory sigma protein, which is required for initiation of transcription • Which is a co-enzyme

9. Bacterial RNA Polymerase

10. Sigma Factors • Binds to the core enzyme, enabling RNA polymerase to recognize and bind to specific sites on DNA, called promoters. • Sigma is the factor responsible for identifying the start site of a gene • Different sigma factors are activated in response to different environmental conditions

11. Promoters • Two key regions : • The –10 box has the sequence 5'-TATAAT-3‘ • located 10 bases upstream of the transcription start site • The –35 box has the sequence 5'-TTGACA-3‘ • located 35 bases upstream of the transcription start site • Sigma identifies the –10 and –35 promoter sites, properly orienting the RNA polymerase core complex for transcription at the gene start site

12. How Transcription Begins

13. How Transcription Begins

14. How Transcription Begins

15. Elongation and Termination • Sigma dissociates from the core enzyme once the initiation phase of transcription is completed • RNA polymerase moves along the DNA template in the 3'  5' direction • Synthesizes RNA in the 5'  3' direction • RNA polymerase encounters a transcription termination signal • Causes the RNA to form a hairpin structure

16. Transcription in Eukaryotes

17. Eukaryotic Polymerases • Contains many polymerases depending on gene type

18. Initiation of Translation • Promoter- specific sequence of DNA where polymerase attaches and transcription begins • Upstream of the gene • Promoters signal the initiation of RNA synthesis • Transcription factors • Help eukaryotic RNA polymerase recognize promoter sequences

19. DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Polypeptide Promoter 5 3 A T A T A A A A T A T T T T 3 5 TATA box Start point Template DNA strand Eukaryotic promoters 1 Transcription factors 5 3 3 5 Several transcription factors 2 Additional transcription factors 3 RNA polymerase II Transcription factors 3 5 5 3 5 RNA transcript Transcription initiation complex Initiation of Transcription • Promoter (TATA box) • Several transcription factors bind to DNA before polymerase • Polymerase binds to transcription factors • Transcription starts

20. Elongation • As RNA polymerase moves along the DNA • It continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides • Enzyme adds at 3’ end • Many polymerases can work on the same strand to produce many copies

21. Termination • Differs in prokaryotes and eukaryotes • Prokaryotes- terminator sequence causes polymerase to detach and mRNA to detach • Eukaryotes- polymerases keep on adding nucleotides but proteins associated with the mRNA cut it free from the growing strand

22. TRANSCRIPTION DNA Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Polypeptide Alteration of mRNA • Cap and Tail • The 5 end receives a modified nucleotide cap • For recognition • The 3 end gets a poly-A tail • Protects RNA from Degradation A modified guanine nucleotide added to the 5 end 50 to 250 adenine nucleotides added to the 3 end Polyadenylation signal Protein-coding segment 5 3 G P P AAA…AAA P AAUAAA Start codon Stop codon Poly-A tail 5 Cap 5 UTR 3 UTR

23. Intron Exon 5 Exon Intron Exon 3 5 Cap Poly-A tail Pre-mRNA TRANSCRIPTION DNA 30 31 104 105 146 1 Pre-mRNA RNA PROCESSING Introns cut out and exons spliced together Coding segment mRNA Ribosome TRANSLATION 5 Cap Poly-A tail mRNA Polypeptide 1 146 3 UTR 3 UTR Alteration of mRNA • RNA splicing- Removes introns and joins exons

24. Introns • The presence off introns • Allows for alternative RNA splicing • Regulatory function • May be the reason humans have so few genes • Proteins often have a modular architecture consisting of discrete structural and functional regions called domains • Different exons can code for the different domains in a protein

25. Gene DNA Exon 1 Exon 2 Intron Exon 3 Intron Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide Introns

26. Eukaryotic vs. Prokaryotic Transcription

27. Translation

28. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C C C G G Anticodon A A A A G G G U G U U U C Codons 5 3 mRNA Translation RNA-directed synthesis of a polypeptide • A cell translates an mRNA message into protein with the help of transfer RNA (tRNA)

29. Bacterial Translation • Ribosomes catalyze translation of the mRNA sequence into protein. • In bacteria, ribosomes begin translation of an mRNA before RNA polymerase has finished transcribing it

30. Eukaryotic Translation • In eukaryotes, mRNAs are synthesized and processed in the nucleus and transported to the cytoplasm for translation by ribosomes

31. Role of Transfer RNA • Each tRNA carries a specific amino acid that is transferred to protein • The addition of amino acids to tRNAs is mediated by aminoacyl tRNA synthetase • Molecules of tRNA are not all identical • Each carries a specific amino acid on one end and an anticodon on the other end • Consists of a single RNA strand that is only about 80 nucleotides long • Is roughly L-shaped

32. Amino acid attachment site 5 3 Hydrogen bonds 3 A Amino acid attachment site C A A G C 5 A 3 5 C G Anticodon C G Anticodon C G U G U A A U U A U C G * G U A C A C A * A U C C * G * U G U G G * G A C C G * C A G * U G * * G A (a) Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.) G C Hydrogen bonds G C U A G * A * A C * U A G A (b) Three-dimensional structure Anticodon tRNA

34. Ribosomes • Ribosomes can be separated into two subunits, the large subunit and the small subunit • tRNAs are found on three sites in ribosomes: • A site is the acceptor site for the aminoacyl-tRNA • P site holds the growing polypeptide chain • E site is where tRNAs no longer bound to an amino acid exit the ribosome

36. Ribosomes • A peptide bond forms between the amino acid on the aminoacyl-tRNA in the A site and the existing polypeptide held on the tRNA in the P site • Polypeptide is transferred to the tRNA in the A site. • The ribosome moves forward to the next codon • tRNA in the E site exits the ribosome

37. Ribosomes • The tRNA in the P site moves into the E site • The tRNA with the polypeptide chain moves from the A site to the P sit • A new aminoacyl-tRNA enters the A site.

38. Amino end of polypeptide 1 Codon recognition. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide E mRNA 3 Ribosome ready for next aminoacyl tRNA P A site site 5 2 GTP GDP 2 site E E P A P A 2 Peptide bond formation. An rRNA molecule of the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site. GDP Translocation. The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site. 3 GTP E P A Ribosomes

39. Initiation of Translation • Translation begins at the AUG start codon • Complementary to a section of one rRNA in the small ribosomal subunit • Once the small ribosomal subunit is bound to the mRNA, the aminoacyl initiator tRNA binds to the AUG sequence • The large subunit binds and completes the initiation complex. The initiator tRNA is located in the P site of the ribosome

40. Initiation

41. Elongation • At the start of elongation, the initiator tRNA is in the P site, and the E and A sites are empty • An aminoacyl tRNA binds to the codon in the A site via complementary base pairing between anticodon and codon, and peptide bond formation occurs • Etc, etc • The ribosome translocates down the mRNA by three nucleotides, and the tRNA attached to the growing protein moves into the P site

42. Polyribosomes • Strings of ribosomes, assemble along an mRNA to increase the rate of protein production

43. Elongation • The three steps in elongation— • arrival of aminoacyl tRNA • peptide bond formation • translocation—repeat down the length of the mRNA.

44. Termination • When the A site encounters a stop codon, a release factor enters the site and catalyzes hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain

45. Termination

46. Post-Translational Modifications • Most proteins go through an extensive series of processing steps before they are ready to go to work in a cell. • Get into proper conformation • Molecular chaperones speed folding of the protein • Small chemical groups may be added to eukaryotic proteins in the rough endoplasmic reticulum

47. Post-Translational Modifications • Some proteins receive a sorting signal to ensure that the molecule will be carried to the correct location in the cell • Some proteins are augmented with sugar or lipid groups that are critical for normal functioning. • Many proteins are altered by enzymes that add or remove a phosphate group • Switch the protein from an inactive state to an active state or vice versa

48. The Molecular Basisof Mutation

49. Mutation • Any change in an organism’s DNA sequence. DNA mutations affect phenotype only when the mutation is expressed • Resulting protein functions abnormally • Not all mutations affect the protein’s ability to function and thus do not generate a phenotype

50. Point Mutation • Change in a single nucleotide • Most common • Can result from errors in DNA replication or from exposure to mutagenic toxins