Transcription and translation

1. Transcription and translation The link between genes and enzymes

2. “Inborn errors of metabolism” • A. Garrod, 1902 • Certain diseases seemed to be inherited • Alkaptonuria due to an enzymatic defect • Beadle and Tatum, 1941- one gene, one enzyme • Studied Neurospora crassa • Nutritional (auxotrophic) mutants • Biosynthetic pathways

3. Beadle and Tatum experiment • Pathway for arginine biosynthesis was known • Used media with defined supplements to identify mutants • One gene, one polypeptide hypothesis defines relationship between DNA and protein

4. The central dogma of molecular biology DNA  RNA  Protein

5. RNA is involved in protein synthesis, too • Messenger RNA- the gene sequence • Ribosomal RNA- structural component of the ribosome • Transfer RNA- interpret mRNA and build the amino acid sequence on the ribosome • In eukaryotes: • Small nuclear RNAs- • SRPs (signal recognition particles) • miRNA (micro-RNA)

6. The triplet code • Four nucleotides • Triplets give 64 possible combinations to code for 20 amino acids • Code is nonoverlapping • One DNA strand serves as the template

7. Transcription: RNA copied from DNA • RNA polymerase uses DNA as a template to make RNA • Template is the “antisense” strand 5’-TACGGTACATTCGTACC ATC T -3’ 3’-ATGCCATGTAAGCATGGTAGA -5’ mRNA:5’- UACGGUACAUUCGUACCAUCU-3’

8. Deciphering the genetic code • 64 possible codons • Experimental requirements: • Cell-free systems with all the necessary enzymes • Synthetic RNAs • Nirenberg, 1961-6: • Worked with different combinations of RNAs to deduce codons • Khorana (overlapped Nirenberg) • Synthesized RNA molecules of defined sequences and analyzed peptides produced • Also synthesized acetyl CoA and the first artificial chromosome (1970)

10. Some exceptions to the code • Genetic code is shared by all organisms • Some variations in mitochondrial, chloroplast and protozoan DNA (ribosomes are different, too) • Processes of transcription and translation are very similar in prokaryotes and eukaryotes, but not identical • Initiation, elongation, termination

11. Properties of RNA polymerase • Two forms: holoenzyme and core polymerase • Need both for accurate initiation of RNA synthesis • Promoter and start site • Accurate termination • Process is simpler in prokaryotes: one polymerase

12. Models of transcription events

13. Operons: transcription and translation coupled in prokaryotes repressor Regulatory promoter operator structural genes region

14. Eukaryotic vs prokaryotic transcription • Eukaryotes have three RNA polymerases, each with their own promoters • RNA polymerase I- rRNA • Probably species-specific • RNA polymerase II- mRNA and snRNAs • Core promoter • RNA polymerase III- tRNA and some small RNAs • Promoter is internal • All work in cell nucleus

15. Eukaryotic initiation complex contains many cofactors Note that transcription factors bind first

16. Elongation and termination of transcription • Multiple molecules of polymerase can transcribe DNA simultaneously • Termination mechanism is different in eukaryotes

17. Another difference: posttranscriptional modification • 5’ cap • Guanine is methylated and linked to 5’ end of transcript • 3’ poly-A tail • Specific cleavage site AAUAA • Poly-A polymerase adds poly-A tail after that site • Splicing of “pre-mRNA”

18. Eukaryotic mRNA is spliced • Introns and exons • Much more human DNA is in introns than exons (exons are 1-1.5% of total) • Splicing involves snRNPs

19. Model for pre-mRNA splicing

20. What are the rules for intron frequency and size? • There are none • Some genes have many introns, some none • Probably accumulated over time • “Exon shuffling” seen among some families of proteins • Alternative splicing seen (remember the codon rule)

21. Overview of translation

22. Structure of transfer RNA (tRNA)

23. tRNA charging ensures that amino acids are positioned correctly

24. tRNA and the ribosome • E site- for tRNA with amino acid already added (exit) • P site- for amino acid being added (peptidyl) • A site for the incoming amino acid (aminoacyl) • This is where peptide bonds are formed (remember primary structure?)

25. Initiation of translation in prokaryotes:note the order of events AUG is start codon; first amino acid is N-formylmethionine Methionine in eukaryotes; also more initiation factors Small subunit binds to 5’-cap instead of a ribosome-binding sequence

26. Elongation cycle Wobble pairing gives flexibility (note orientation of anticodon)

27. Termination triggered by a stop codon

28. In eukaryotes, many proteins are processed in the ER • Prokaryotes have this mechanism, too • Secreted proteins require it

29. Summary: gene expression in bacteria

30. Gene expression in eukaryotes

31. Mutations occur in DNA

32. Does mutation affect phenotype? • Triplet repeats- add to reading frame but do not shorten it • Might be in coding or noncoding region • Sometimes they are big enough to see in a karyotype

33. Chromosomal mutations can have drastic effects • Deletions • Duplications • Can lead to “gene families” and pseudogenes • Inversions • Translocations • Can be fatal, can be inherited; tend to arise in single cells (somatic mutation)

34. Summary • Genes specify polypeptides • Transcription makes RNA copies of DNA • Translation involves mRNA, tRNA, and rRNA in protein synthesis • Processes are similar in prokaryotes and eukaryotes, but there are significant differences • The genetic code is nearly universal • Mutations alter DNA and can alter genes and proteins • Evolution arises from mutation