Molecular Biology of Gene Expression

1. Molecular Biology of Gene Expression Genetics Spring 2014

2. Outline

3. The Central Dogma of Molecular Biology follows these steps excepts for structural RNAs (tRNA, rRNA and other small RNAs that are not translated). This model displays colinearitywhere sequence in DNA and sequence in polypeptides are colinear (5’ end of gene  5’ end of mRNA  NH2 end of polypeptide). DNA transcription RNA translation Protein

4. Transcription - Overview • Precursorsare the 4 ribonucleosides 5’-triphosphate (rNTPs) ATP, UTP, CTP, GTP. • RNA polymerase (RNA Pol) binds special DNA sequence (promoters) and starts making RNA without the need of primer. • RNA is synthesized 5’ -> 3’; linkage of nucleotides phosphodiester bond. • Linear order of RNA bases is determined by DNA sequence (complementary base pairs), leading to a primary transcripts. • Each RNA molecule is transcribed from one strand of DNA (template strand) from its 3’ end; a transcription bubble is formed briefly.

5. Transcription – Different types of Polymerase All polymerases are holoenzymesformed of an apoenzyme (multiple sub-units) and several cofactors. • Bacteria have only one RNA polymerase that transcribes all genes. • 6 subunits; sigma factor (s) recognizes promoter • very processive (~ 104 nucleotides before stop) • very fast, ~ 70 nucleotides/sec • Eukaryotes have several RNA polymerases: • RNA polymerase I - rRNA • RNA polymerase II - mRNA and certain small RNAs • RNA polymerase III - tRNAs, 5S rRNA Structure of the Pol II holoenzyme

6. Transcription – Promoter Recognition E. coli promoters have conserved or consensussequences as -35 and -10 nt where start of transcription is +1. Eukaryote promoters are more complex although the TATA box promoter is conserved between bacteria and humans. In E. coli, the sigma factor allows for proper binding of RNA pol to the promoters. In eukaryotes, more than 26 transcription factors are necessary.

7. In eukaryotes, the formation of the transcriptomeallows for fine-tuning gene regulation. Promoters, silencersand enhancers respectively bind to basic transcription factors and regulatory transcription factors repressorsand activators.

8. Transcription– Chain Termination In prokaryotes, self-termination (intrinsic termination) sequences contain hairpin and a U-rich region or a terminator protein rho. In eukaryotes, the intrinsic termination are more complex. transcription–termination region of tryptophan-synthesizing genes in E. coli 3' terminus of transcript can form stem-and-loop structure

9. Only one DNA strand is transcribed for a gene, however different genes can be transcribed on different strands. The primary transcripts (pre-mRNAs) need to be processed to generate a mRNAs (not as degradable) used as templates in translation.

10. RNA Processing in Eukaryotes – The steps • 5' capping - addition of 7-methylguanosine (MeG) in 5' to 5' linkage at the 5' end of mRNA. • 3' cleavage (trimming) after RNA pol II passes specific region. • 3’ polyadenylation - addition of ~200 nt polyA tail by special polyA polymerase. • splicing - removal of introns and joining of exons.

11. Transcription and processing are coupled

12. RNA Processing in Eukaryotes – RNA Splicing Introns have 5' GU and 3' AG splice sites and internal branch site A

13. Splice site mutations may result in retention of an intron, and abnormal proteins. • Mutations within an intron are often less deleterious.

14. Translation - Overview • mRNA - the nucleotide coding sequence that determines amino acid sequence. • Ribosomes - responsible for protein synthesis; rRNA is catalytic component. • tRNA - adaptor molecules that decode nucleotide sequence to amino acid sequence. • Aminoacyl tRNA synthetase - attaches an amino acid to tRNA “charges the tRNA” (a second code). • Initiation, elongation, and release factors (specialized proteins that aid in each stage of translation).

15. Amino Acids, Polypeptides, and Proteins • Each 20 amino acids contains an asymmetric carbon (Ca) linked to 4 different groups: • Hydrogen • Amino group • Carboxyl group • R group (side chain that give diversity of chemical properties)) Amino acids are linked together through peptide bonds (condensation reaction) to forms polypeptidesthat in turn form functional proteins after secondary, tertiary or quaternary structure formation.

17. Translation– The steps Initiationof protein synthesis is complex and involves many special proteins Eukaryotes have 5’ CAP, ribosome scanning to find AUG

18. Elongation is a repeated cycle of 3 processesNew tRNA arrives, H-bonds to mRNA;new peptide bond as growing chain is handed to next aa;ribosome moves to next codon.

19. Translation terminationoccurs when stop codon is reachedSpecial release factor proteins assist

20. Alternative pathways in protein foldingChaperones help proteins fold correctly

21. Complex Translation Units In bacteria, transcription and translation are coupled;polysomesare multiple ribosomes on 1 mRNA. In eukaryotes, transcription takes place in the nucleus and translation occurs in the cytoplasm. Visualization of transcription, translation in bacteria

22. The Genetic Code Deletion and insertion mutations can suppress each other.

23. Review Problems

24. 10-23:Two E. coli genes, A and B are known from mapping experiments to be very close to each other. A deletion mutation is isolated that eliminates the activity of both A and B. Neither the A nor the B protein can be found in the mutant, but a novel protein is isolated in which the amino-terminal 30 amino acids are identical to those of the B gene product, and the carboxy-terminal 30 amino acids are identical to those of the A gene product. With regard to the 5’ to 3’ orientation of the non -transcribed DNA strand, is the order of the genes AB or BA? Can you make any inference about the number of bases deleted in the coding region?

25. 10-30:An organism is discovered in which the promoter sequence in the template strand is 3’-TTTT-5’, the transcript begins with the first nucleotide following the promoter sequence, and transcription terminates immediately prior to the sequence 3’-GGGGGG-5’ in the template strand. The primary transcript is capped and used directly for the mRNA, and translation is initiated by scanning from the 5’ end. What mRNA sequence would be transcribed form the DNA sequence below? What polypeptide chain would result? 3’-TTTTTATGGTACAGTTTGTCGCATACCATCGTCACGGGGGG-5’

26. An organism is discovered whose RNA polymerase II has the following properties: 1. The DNA sequence in the template strand 3’–TATAATA–5’ serves as the promoter. 2. Transcription begins at the nucleotide immediately following the promoter. 3. Transcription continues until the transcript includes the sequence 5’GGGGG 3’, at which point the polymerase switches to the other DNA strand and continues transcribing (still adding nucleotides only to the 3’ end of the growing chain) 4. Transcription terminates immediately after transcribing 5’–TATATA–3’ from the template strand. If the primary transcript is immediately capped and used as the mRNA, and translation is initiated by scanning from the 5’ end: (a) What mRNA would be produced from the DNA sequence shown here? (b) What polypeptide chain results? 5’-CCGTATATATTATGATCAATATGCATGCTCTCGGGGGTCACACT-3’ 3’-GGCATATATAATACTAGTTATACGTACGAGAGCCCCCAGTGTGA-5’