Gene Expression in molecular biology.pptx

Gives details on how Gene's are expressed in the DNA via different CELLULAR processes, and gives analysis of different Gene's which are affected by factors

1. Transcription

2. Transcription The copying of the sequence of the template strand of the gene into a complementary RNA transcript (mRNA) The beginning of gene expression Transcription and translation are coupled in bacteria In eukaryotes, mRNA and protein synthesis are separated between two cellular compartments

3. Transcription and translation coupled in prokaryotes

4. Transcription and translation uncoupled in eukaryotes Regulation at each level

5. Sense and Antisense strands A DNA sequence is read in triplets using the antisense ( non-coding ) strand; Called the template strand , directs the synthesis of RNA via complementary base-pairing The other non-template strand is the sense ( coding ) strand; Bears the same sequence as the mRNA (except for possessing U instead of T)

6. Sense and antisense strands

7. Promoters: in bacteria they have two distinct “Consensus” Sequences A promoters is a sequence to which RNA polymerase binds to begin transcription

8. Structure of bacterial RNA pol Comprised of; Core Enzyme , and A transcription factor called the Sigma factor, δ Together, the core enzyme and δ form a functional enzyme complex called the “ Holoenzyme ”

9. The core enzyme, cont’d The core enzyme catalyzes polymerization It is conserved from bacteria to humans Has high affinity for most DNA; in absence of δ it initiates synthesis anywhere on a DNA template in vitro The δ is responsible for decreasing the non-specific binding affinity of RNA pol

10. The sigma factor ( δ ) Recognizes the promoter The -35 and -10 sequences are required for recognition -10 region is responsible for initial melting of the template strand There several δ per species

11. E. coli sigma factors

12. Types of DNA-dependent RNA polymerases RNA polymerases catalyze RNA synthesis using DNA as a Template; they initiate RNA synthesis de novo (in contrast to DNA pol) Sites for antibiotic attack (RIF, etc.)

13. The mechanism of transcription Occurs in three stages Initiation Elongation Termination

14. Initiation Further divided into three stages; Formation of a closed promoter complex The DNA remains DS and the complex is reversible Formation of an open promoter complex Melting occurs to expose the template strand AT rich -10 region and negative supercoiling helps Irreversible; polymerization is initiated Promoter clearance There is a staged disruption of δ -core enzyme interaction

15. Elongation Transcription bubble is formed as RNA pol winds and unwinds strands The catalytic site of the RNA pol has both; A Substrate Binding Sub-site at which the incoming NTP is bound to the pol and to the complementary nt residue of the template And a Product Binding Sub-site , at which the 3’-terminus of the growing RNA chain is positioned NTP and a phosphodiester bond forms with the 3’-OH of the last nt in the RNA chain Transcription also proceeds in 5’ – 3’

16. Transcription elongation

17. Termination The RNA pol moves down the DNA until a stop signal or terminator sequence is reached Two types of terminators; Rho dependent Rho independent All recognize inverted repeats that form stem-loop structures

18. Gene expression regulation Except the house keeping genes, not all genes are expressed all at once Certain gene products (proteins) regulate / control expression of the genes The “Operon” model by Jacob & Monod led to the discovery of mRNA Read the Lac, Try and Arabinose operons Signal transduction: regulates gene expression mostly in eukaryotes Quorum Sensing / Cell-Cell Signalling : regulates gene expression in bacteria and some unicellular protozoa (African Trypanosomes)

19. Translation

20. Translation The process in which the RNA nucleotide sequence is converted into an amino acid sequence of a protein The term denotes that the info in the language of nucleotides is copied (translated) into another language of amino acids Translation occurs at the ribosomes, and understanding their structures is key

21. Two functions of ribosomes Decoding the genetic code in the mRNA Catalyzing the formation of the peptide bonds between amino acids resulting in a polypeptide chain In essence, a ribosome is an enzyme, A Polypeptide Polymerase

22. Structural significance of ribosomes Ribosomes have two subunits, large and small Prokaryotic: 70S , comprising of 50S & 30S Eukaryotic: 80S , comprising of 60S & 40S The Peptidyl Transferase center and the catalytic site are in the large s/unit (60S / 50S) The small subunit serves as the assembly guide for factors needed in protein synthesis; decoding the mRNA also occurs on the small subunit (40S / 30S)

23. Recap; Components of Ribosomes

24. Ribosome binding sites Ribosomes have 3 tRNA -binding sites The Acceptor site (A) The Peptidyl site (P) The Exit site (E) Each of the above is occupied in succession by a particular tRNA during protein synthesis cycle

25. A-site: occupied by aminoacyl- tRNA , i.e. the charged tRNA P-site: occupied by peptidyl- tRNA , i.e. the tRNA carrying the growing peptide chain. The P-site is also referred to as the Puromycin sensitive site. Puromycin is an antibiotic which shows similarities with a part of aminoacyl- tRNA . E-site: the ribosomal site harboring decylated tRNA on transit out from the ribosome.

26. Ribosome binding sites P site A site E site 40S/30S (small subunit) 60S/50S (large subunit) Anticodon

27. tRNAs bridge the large and small s/units; With the anticodon arm of tRNA pointing towards the small s/unit for decoding And the acceptor arm of tRNA pointing into the large s/unit for peptidyl transferase Ribosomes function in the cytoplasm, but their assembly occurs in the nucleolus

28. The nucleolus and ribosome biogenesis The nucleolus is the site of rRNA synthesis and ribosome assembly Except for 5S rRNA , rRNA genes are transcribed by RNA pol I into one long precursor rRNA 5S rRNA is transcribed from a separate gene by RNA pol II Eukaryotic large and small ribosomal s/units are assembled in the nucleolus before export to the cytoplasm

29. Aminoacyl-tRNA synthetases The fidelity of translation is dependent on the accuracy of two processes; Codon-Anticodon recognition Aminoacyl-tRNA synthesis Aminoacyl-tRNAs are synthesized by the 3’-Esterification of tRNAs with the appropriate amino acid; Aminoacyl-tRNA synthetases catalyze the reactions The uncharged tRNA is aminoacylated to generate a charged tRNA , which then interacts with the elongation factor ( eEF )

30. Aminoacyl-tRNA charging Aminoacyl- tRNA synthetases attach amino acids to tRNAs in two enzymatic steps; Amino acid reacts with ATP to become adenylated (addition of AMP). Amino acid is attached by a high energy ester bond between the carbonyl group of the amino acid and the phosphoryl group of AMP AMP is released and the amino acid is transferred to the 3’-end of tRNA to form charged tRNA Specific aminoacyl-tRNA synthetases are denoted by their 3 letter amino acid designation Met RS ; Methyl- tRNA synthetase ( Enzyme ) tRNA met ; Uncharged tRNA specific for Methionine Met - tRNA ; tRNA aminoacylated with Methionine ( Charged tRNA ) Each of the 20 amino acids has a specific aminoacyl-tRNA synthetase

31. Activated/charged tRNA Two steps in aminoacyl tRNA charging

32. The mechanism of translation Also occurs in three stages; Initiation Elongation Termination Each step involves multiple factors and energy from GTP hydrolysis

33. Initiation The most complex, most tightly controlled stage The ribosome is assembled at the Initiation Codon in the mRNA with a Methionyl Initiator tRNA bound to its P site Initiation further subdivided into; Formation of Ternary Complex and Loading onto the 40S s/unit Loading the mRNA on the 40S s/unit Scanning the start codon recognition Joining of the 40S and 60S s/units to form the functional 80S s/unit

34. Ternary complex formation and loading onto the 40S s/unit Assembly of the ternary complex is the 1 st step in the initiation pathway Ternary complex is comprised of eIF2 , GTP , and the amino acid-charged initiator tRNA ( Met- tRNA ) The complex binds to the 40S s/unit to form a 43S complex

35. Loading the mRNA on the 40S s/unit In bacteria (coupled transcription/translation) soon as Shine- Dalgano emerges from the transcriptional apparatus, it is bound by the 30 small s/unit Eukaryotic mRNA to be translated is fully processed; Spliced , 5’-capped , poly-A tailed The 5’-end of mRNA is identified by the m 7 GpppN cap IFs associate with 5’-cap, unwind 2 o and 3 o structures and remove RNA binding proteins Other IFs associate with poly A binding protein bound to the 3’-poly A tail Poly A tail recruits the 43S complex to the mRNA and when bound with PABP, signals translation Together, the 5’ and 3’ IF complexes work to load the mRNA onto the 43S complex

36. Scanning and AUG recognition Once the mRNA is loaded, the 43S complex scans along the message from 5’ to 3’ looking for AUG start codon Once the 43S complex encounters an AUG codon (usually 1 st AUG) embedded in the kozak consensus sequence; a stable 48S complex is formed Upon arrival at AUG, codon-anticodon interaction occurs by complementary bp with an antiparallel orientation between tRNA and mRNA

37. Joining of the 40S and 60S s/units Initiation involves two GTP hydrolysis events; One catalyzed by eIF2 upon AUG recognition Another at the end of the pathway, after 80S complex formation

38. Translation initiation

39. Elongation Similar in prokaryotes and eukaryotes Occurs rapidly; aminoacyl tRNA enter the A site where decoding occurs If they are the correct ( Cognate ) tRNAs , the ribosome catalyzes formation of a peptide bond between the incoming aa and the growing polypeptide chain After the tRNAs and the mRNAs are translocated such that the next codon is moved to the A site, the process is repeated

40. Elongation, details Peptide chain elongation begins with a peptidyl-tRNA in P site next to a vacant A site An aminoacyl-tRNA is carried to the A site as part of a ternary complex with GTP and eEF1A Cognate (correct) codon-anticodon bp causes 3 bases in the 18S rRNA to swing out and interact with the resulting mRNA- tRNA duplex This activates the GTPase activity of eEF1A eEF1A GDP releases the aminoacyl-tRNA into the A site in a form that can continue with peptide bond formation

41. Peptide bond formation and translocation The ribosomal peptidyl transferase center catalyzes formation of a peptide bond between the incoming amino acid and the peptidyl-tRNA The resulting deacylated tRNA is moved into the E site of the large s/unit, the peptidyl-tRNA is moved into the P site, and the mRNA moves by 3 nt to place the next codon of mRNA into the A site Translocation is mediated by eEF2 and requires GTP

42. The 23S and 28S rRNAs are catalytic Peptide bond formation is catalyzed by 23S / 28S rRNAs , the ribozymes Discovered in 2000, proving that ribosomal RNA is catalytic Prior to the year 2000, only proteins were thought to be the enzymes (biological catalysts)!

43. Termination The ribosomal peptidyl transferase center is responsible for two fundamental reactions; Peptide bond formation, and Nascent peptide release during elongation and termination phases of protein synthesis Translation termination occurs in response to presence of a stop codon ( UAG, UAA, UGA ) at the A site The end result is the release of the completed polypeptide following hydrolysis of the ester bond linking the polypeptide to the P site tRNA Termination requires two release factors; Class I RFs decode stop codons Class II RFs are GTPase