Chapter 18

1. Chapter 18 Gene Expression and Protein Synthesis

2. Introduction • The central dogma of molecular biology • information contained in DNA molecules is expressed in the structure of proteins • gene expression is the turning on or activation of a gene

3. Transcription • Transcription: the process by which RNA is synthesized from a DNA template • transcription starts when the DNA double helix begins to unwind near the gene to be transcribed (helicase) • only one strand of the DNA is transcribed (template strand) • ribonucleotides assemble along the unwound DNA strand in a complementary sequence • enzymes called polymerases(poly) catalyze transcription: Pol I for rRNA formation, Pol II for mRNA formation, and Pol III for tRNA formation

4. Transcription

5. Transcription • A eukaryotic gene has two parts • a structural gene that is transcribed into RNA; the structural gene is made of exons and introns • a regulatory gene that controls transcription; the regulatory gene is not transcribed but has control elements, one of which is the promoter • a promoter is unique to each gene • there is always a sequence of bases on the DNA strand called an initiation signal • promoters also contain consensus sequences such as the TATA box in which the two nucleotides T and A are repeated many times

6. Transcription • a TATA box lies approximately 26 base pairs upstream • all three RNA polymerases interact with their promoter regions via transcription factors that are binding proteins • after initiation, RNA polymerase zips up the complementary bases in a process called elongation • elongation is in the 5’ -> 3’ direction • at the end of each gene is a termination sequence that tells the enzyme “stop the transcription.”

7. Transcription • Pol II has two different forms • at its C-terminal domain, it has Ser and Thr repeats that can be phosphorylated • when Pol II starts initiation, it is unphosphorylated • upon phosphorylation, it catalyzes elongation • after termination of the transcription, it is dephosphorylated by a phosphatase • in this manner, Pol II is constantly recycled between its initiation and elongation roles

8. Transcription • The RNA products of transcription are not necessarily functional RNAs • they are made functional by post-transcription modification • transcribed mRNA is capped at both ends • the 5’ end acquires a methylated guanine • the 3’ end acquires a polyA tail that may contain from 100 to 200 adenine residues • once the two ends are capped, the introns are spliced out • tRNA is similarly trimmed, capped, and methylated • functional rRNA also undergoes post-transcription methylation

9. RNA in Translation • mRNA, rRNA, and tRNA all participate in translation • protein synthesis takes place on ribosomes • a ribosome dissociates into a larger and a smaller body • in higher organisms, the larger body is called a 60S ribosome; the smaller body is called a 40S ribosome • the 5’ end of the mature mRNA is bonded to the 40S ribosome and this unit then joined to the 60S ribosome • together the 40S and 60S ribosomes form a unit on which mRNA is stretched out • triplets of bases on mRNA are called codons • the 20 amino acids are then brought to the mRNA-ribosome complex, each amino acid by its own particular tRNA

10. tRNA • each tRNA is specific for only one amino acid • each cell carries at least 20 specific enzymes (synthetases), each specific for one amino acid • each enzyme recognizes only one tRNA • the enzyme bonds the activated amino acid to the 3’ terminal -OH group of the tRNA by an ester bond • at the opposite end of the tRNA molecule is a codon recognition site • the codon recognition site is a sequence of three bases called an anticodon • this triplet of bases aligns itself in a complementary fashion to the codon triplet on mRNA

11. tRNA • The three-dimensional structure of a tRNA

12. The Genetic Code • Assignments of triplets is based on several types of experiments • one of these used synthetic mRNA • if mRNA is polyU, polyPhe is formed; the triplet UUU, therefore, must code for Phe • if mRNA is poly ---ACACAC---, poly(Thr-His) is formed; ACA must code for Thr, and CAC for His • By 1967, the genetic code was broken

13. The Genetic Code

14. Features of the Code • all 64 codons have been assigned • 61 code for amino acids • 3 (UAA, UAG, and UGA) serve as termination signals • AUG codes for Met but also serves as an initiation signal • only Trp and Met have one codon each • more than one triplet can code for the same amino acid; Leu, Ser, and Arg, for example, are each coded for by six triplets • the third base is irrelevant for Leu, Val, Ser, Pro, Thr, Ala, Gly, and Arg

15. Features of the Code • for the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies. Gly, for example, is coded for by GGA, GGG, GGC, and GGU • the code is almost universal: it the same in viruses, prokaryotes, and eukaryotes; the only exceptions are some codons in mitochondria

16. Reading the Genetic Code • Suppose we want to determine the amino acids coded for in the following section of a mRNA 5’—CCU —AGC—GGA—CUU—3’ • According to the genetic code, the amino acids for these codons are: CCU = Proline AGC = Serine GGA = Glycine CUU = Leucine • The mRNA section codes for the amino acid sequence of Pro—Ser—Gly—Leu

17. Translation • Translation: the process whereby a base sequence of mRNA is used to create a protein • There are four major stages in protein synthesis • activation • initiation • elongation • termination

18. S t a g e M o l e c u l a r C o m p o n e n t s A c t i v a t i o n A m i n o a c i d s , A T P , t R N A , a m i n o a cyl c - t R N A syn s n theta t se I n i t i a t i o n t R N A f M e t , 4 0 S r i b o s o m e , i n i t i a t i o n f a c t o r p r o t e i n s , m R N A , 6 0 S r i b o s o m e E l o n g a t i o n 4 0 S a n d 6 0 S r i b o s o m e s , t R N A , e l o n g a t i o n f a c t o r p r o t e i n s , m R N A , p e p t i d y l t r a n s f e r a s s T e r m i n a t i o n R e l e a s i n g f a c t o r p r o t e i n s , t R N A m R N A , 4 0 S a n d 6 0 S r i b o s o m e s Protein Synthesis • Molecular components of reactions at four stages of protein synthesis

19. Amino Acid Activation • Requires • amino acids • tRNAs • aminoacyl-tRNA synthetases • ATP, Mg2+ • Formation on an aminoacyl-tRNA

20. Amino Acid Activation • This two-stage reaction allows selectivity at two levels • the amino acid: the aminoacyl-AMP remains bound to the enzyme and binding of the correct amino acid is verified by an editing site on the tRNA synthetase • tRNA: there are specific binding sites on tRNAs that are recognized by aminoacyl-tRNA synthetases Recognition by an enzyme (synthetase)of its proper tRNA and amino acid is often referred to the second genetic code.

21. Chain Initiation • Initiation consists of three stages • forming the pre-initiation complex: tRNAfMet attaches to small ribosomal subunit • migration to mRNA: the pre-initiation complex binds to mRNA • forming the full ribosomal complex: the large ribosomal subunit joins the small subunit. The growing peptide chain will bind to the P site of the ribosome

22. Elongation • Binding to the A site: A tRNA, whose anticodon matches the next codon on the mRNA, binds to the vacant A site. • Forming the 1st peptide bond: On the A site, the alanine is linked to the fMet in a peptide bond by the enzyme peptidyl transferase. The empty tRNA remains on the P site. • Translocation: the whole ribosome moves one codon along the mRNA, while the dipeptide is simultaneously translocated from the A site to the P site. The empty tRNA is moved to the E site. When the cycle repeats the tRNA is ejected from the ribosome.

23. Elongation (cont’d) • Forming the 2nd peptide bond: A new tRNA, whose anticodon matches the next mRNA codon, enters the now empty A site. The peptidyl transferase creates a peptide bond with the new amino acid, moving the dipeptide from the P site to the A site and forming a tripeptide. • These elongation steps are repeated until the last amino acid chain is attached.

24. Elongation

25. Elongation

26. Elongation

27. Termination • After the final translocation, the next codon reads “stop” (UAA, UAG, or UGA are termination codons). No more amino acids can be added at this point. • Releasing factors then cleave the polypeptide chain from the last tRNA. At the end, the whole mRNA is released from the ribosome.

28. Gene Regulation • Gene regulation: the various methods used by organisms to control which genes will be expressed and when. • some regulations operate at the transcriptional level (DNA -> RNA) • others operate at the translational level (mRNA -> protein)

29. Transcriptional Level • In eukaryotes, transcription is regulated by three elements: promoters, enhancers, and response elements • Promoters • located adjacent to the transcription site • they are defined by an initiator and conserved sequences such as TATA box or GC box • different transcription factors bind to different modules of the promoter • transcription factors allow the rate of synthesis of mRNA (and from there the target protein) to vary by a factor of up to a million

30. Promoters • transcription factors find their targeted sites by twisting their protein chains so that a certain amino acid sequence is present at the surface • one such conformational twist is provided by metal-binding fingers (next slide) • two other prominent transcription factor conformations are the helix-turn-helix and the leucine zipper • transcription factors also possess repressors, which reduce the rate of transcription

31. Promoters • A schematic of a metal-binding finger

32. Enhancers • Enhancers speed up transcription • they may be several thousand nucleotides away from the transcription site; a loop in DNA brings the enhancer to the initiation site

33. Response Elements • Response elements are activated by their transcription factors in response to an outside stimulus • the stimulus may be heat shock, heavy metal toxicity, or a hormonal signal • the response element of steroids is in front of and about 250 base pairs upstream from the starting point of transcription

34. Translational Level • During translation, there are a number of mechanisms that ensure quality control • AARS control • each amino acid must attach to the proper tRNA • enzymes called aminoacyl-tRNA synthetases (AARS) catalyze this attachment • each AARS recognizes its tRNA by specific nucleotide sequences • further, the active site of each AARS has two sieving portions (one that prevents larger amino acids from entering, and one that blocks smaller amino acids)

35. Translational Level • Termination control • the stop codons must be recognized by release factors • the release factor combines with GTP and binds to the ribosomal A site when that site is occupied by the termination codon • Post-translational control • in most proteins, the Met at the N-terminal end is removed by Met-aminopeptidase • certain proteins called chaperones help newly synthesized proteins to fold properly • if rescue by chaperones fails, proteasomes may degrade the misfolded protein

36. Mutations and Mutagens • Mutation: a heritable change in the base sequence of DNA • it is estimated that, on average, there is one copying error for every 1010 bases (1 in 10 billion) • mutations can occur during replication • base errors can also occur during transcription in protein synthesis (a nonheritable error) • consider the mRNA codons for Val, which are GUA, GUG, GUC, and GUU • if the original codon in DNA is GTA and it is misspelled GTG in the copy, it will be transcribed onto mRNA as GUG which also codes for Val • other errors in replication may lead to a change in protein structure and be very harmful

37. Mutations and Mutagens • Mutagen: a chemical that causes a base change in DNA (e.g. carcinogens) • Many changes in base sequence caused by radiation and mutagens do not become mutations because cells have repair mechanisms called nucleotide excision repair (NER) • NER can prevent mutations by cutting out damaged areas and resynthesizing them • Not all mutations are harmful • certain ones may be beneficial because they enhance the survival rate of the species

38. Recombinant DNA • Recombinant DNA: DNA from two sources that have been combined into one molecule • One example of the technique begins with plasmids found in the cells of Escherichia coli • plasmid: a small, circular, double-stranded DNA molecule of bacterial origin • a class of enzymes called restriction endonucleases cleave DNA at specific locations • one, for example, may be specific for cleavage of the bond between A-G in the sequence -CTTAAAG-

39. Recombinant DNA • in this example “B ” stands for bacterial gene, and “H for human gene • the DNA is now double-stranded with two “sticky ends”, each with free bases that can pair with a complementary section of DNA • next, we cut a human gene with the same restriction endonuclease; for example the gene for human insulin

40. Recombinant DNA • the human gene is now spliced into the plasmid by the enzyme DNA ligase • splicing takes place at both ends of the human gene and the plasmid is once again circular • the modified plasmid is then put back into the bacterial cell where it replicates naturally every time the cell divides • these cells now manufacture the human protein, in our example human insulin, by transcription and translation

41. Recombinant DNA

42. Recombinant DNA

43. Protein Synthesis End Chapter 18