1. Transcription

2. Central Dogma from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates

3. Types of RNA messenger RNA (mRNA). This will later be translated into a polypeptide. ribosomal RNA (rRNA). This will be used in the building of ribosomes transfer RNA (tRNA). RNA molecules that carry amino acids to the growing polypeptide. small nuclear RNA (snRNA). DNA transcription of the genes for mRNA, rRNA, and tRNA produces large precursor molecules ("primary transcripts") that must be processed within the nucleus to produce the functional molecules for export to the cytosol. Some of these processing steps are mediated by snRNAs. small nucleolar RNA (snoRNA). These RNAs help process ribosomal RNA (rRNA) molecules.

4. Ribosomal RNA (rRNA) There are 4 kinds. In eukaryotes, these are 18S rRNA. One of these molecules, along with some 30 different protein molecules, is used to make the small subunit of the ribosome. 28S, 5.8S, and 5S rRNA. One each of these molecules, along with some 45 different proteins, are used to make the large subunit of the ribosome.

5. Transfer RNA (tRNA) There are 32 different kinds of tRNA in a typical eukaryotic cell. each is the product of a separate gene many of the bases in the chain pair with each other forming sections of double helix. the unpaired regions form 3 loops each kind of tRNA carries (at its 3' end) one of the 20 amino acids (thus most amino acids have more than one tRNA responsible for them) at one loop, 3 unpaired bases form an anticodon

6. Messenger RNA (mRNA) Messenger RNA comes in a wide range of sizes reflecting the size of the polypeptide it encodes. Most cells produce small amounts of thousands of different mRNA molecules, each to be translated into a peptide needed by the cell. Many mRNAs are common to most cells, encoding "housekeeping" proteins needed by all cells (e.g. the enzymes of glycolysis). Other mRNAs are specific for only certain types of cells. These encode proteins needed for the function of that particular cell (e.g., the mRNA for hemoglobin in the precursors of red blood cells).

7. Transcription from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates

8. Transcription in Prokaryotes Requires a promoter region, with which RNA polymerase interact. The RNA-coding sequence is the DNA that is to be transcribed and later translated. Requires a terminator, downstream of the end of the RNA-coding sequence.

9. Promoter Specific sequences are critical for the recognition by the polymerase. Generally found at –35 and –10; that is they are centered at 35 and 10 bp upstream of +1, transcription start site. Consensus sequence for –35 region (-35 box) is 5’-TTGACA-3’ Consensus sequence for –10 region (Pribnow box) is 5’-TATAAT-3’.

10. RNA Polymerase A form of RNA polymerase called holoenzyme (Complete enzyme) must bind to the promoter. Holoenzyme consists of the the core enzyme (made up of four polypeptides) bound with another polypeptide called the sigma factor, necessary to recognize the –35 and –10 regions of the promoter. Without the sigma factor, the core enzyme doesn’t start transcription efficiently.

11. Binding to the promoter Binds in two steps –Binds loosely to the –35 box while DNA is double stranded. –Binds more tightly to DNA as DNA untwists for about 17 bp centered around the –10 box.

12. Promoter sequences and regulation of transcriptional rate Promoters differ in their sequences slightly, so the binding efficiency of the promoter varies. A –10 region sequence of 5’-GATACT-3’ has a lower rate of transcription initiation than 5’-TATAAT-3’.

13. Sigma factor and transcriptional regulation There are different sigma factors; sigma 70 is the most common sigma32 is used for conditions of heat shock; sequences that are recognized by sigma32 has CCCCC at –39 and TATAAATA at –15.

14. Elongation Transcription bubble forms. Once 8 or 9 RNA nucleotides are linked together, the sigma factor disassociates from the RNA polymerase core enzyme. Core enzyme completes the transcription. As it moves, it untwists the DNA double helix producing torsion, DNA double helix reforms behind the enzyme. Within the untwisted region a temporary RNA- DNA hybrid is formed, the rest of the RNA is displaced away from the DNA.

15. Termination Requires terminator sequences. –Rho-independent termination. These sequences consists of sequences with a two-fold symmetry that makes a hairpin loop, followed by 4-8 AT basepairs, leading to termination when encountered. –Rho-dependent termination. These sequences lack AT string and many cannot form hairpin structures. Rho factor (a protein) is needed to recognize a specific sequence and face the polymerase by moving along the DNA using ATP.

19. Transcription in Prokaryotes Enzyme: RNA polymerase Promoter is the DNA sequence where RNA polymerase binds to initiate transcription

20. Transcription in E.Coli

21. The Operon Model Groups of genes coding for related proteins are arranged in units known as operons. An operon consists of an operator, promoter, regulator, and structural genes.

22. The Operon Model The regulator gene codes for a repressor protein that binds to the operator, obstructing the promoter (thus, transcription) of the structural genes. The regulator does not have to be adjacent to other genes in the operon. If the repressor protein is removed, transcription may occur.

23. Eukaryotic Transcription

24. RNA Processing: pre-mRNA -> mRNA Synthesis of the cap. This is a stretch of three modified nucleotides (7methylguanosine) attached to the 5' end of the pre-mRNA. This structure aligns mRNAs on the ribosome during translation

25. Capping

26. RNA Processing: pre-mRNA -> mRNA Synthesis of the poly(A) tail. This is a stretch of adenine nucleotides attached to the 3' end of the pre-mRNA. This regulates both translation and mRNA stability. It is also important in early development.

27. Polyadenylation

28. RNA Processing: pre-mRNA -> mRNA Splicing. Step-by-step removal of introns present in the pre-mRNA and splicing of the remaining exons.

29. Splicing

30. Three critical sequence elements of pre- mRNA: Sequences at the 5’ splice site sequences at the 3’ splice site sequences within the intron at the branch point

31. Spliceosomes Composed of proteins and RNAs. The RNA components of the spliceosome are small nuclear RNAs (snRNAs) called, U1, U2, U4, U5, and U6. These range between 50 to 200 nucleotides complexed with six to ten protein molecules to form small nuclear ribonucleoprotein particles (snRNPs).

32. Self-splicing Some RNAs are capable of self-splicing. They can catalyze the removal of their own introns in the absence of other RNA or proteins. 28S RNA of the protozoan Tetrahymena. Self-splicing is catalyzed by the intron itself, which acts as a ribozyme.

33. Alternative splicing

34. The RNA polymerases RNA polymerase I (Pol I). Located in nucleolus. It transcribes the rRNA genes for the precursor of the 28S, 18S, and 5.8S molecules (and is the busiest of the RNA polymerases). S values are derived from the rate at which the rRNA molecules sediment during sucrose gradient centrifugation.

35. The RNA polymerases RNA polymerase II (Pol II). Found in nucleoplasm. It transcribes the mRNA and snRNA genes. RNA polymerase III (Pol III). Found in nucleoplasm. It transcribes the 5S rRNA genes and all the tRNA genes.

36. mRNA transcription by RNA PolII RNA polyII produces a precursor mRNA (pre- mRNA). Promoters contain basal promoter elements and promoter proximal elements, having general activity. –Basal promoter elements: TATA box is located at about position –25, and a pyrimidine-rich sequence near the transcription start site is called the initiator element (Inr). TATA box consensus is TATAAAA. –Promoter proximal elements: Located upstream of TATA box, about 50-200 nucleotides from the start of transcription. Examples are CAAT (‘cat’) box, which is located at about –75; GC box with a consensus of GGGCGG, located at about –90.

37. Transcription Factors Basal transcriptional factors are the proteins that are required for the function of RNA polymerase II They bind to the promoter area, TATA Box General Transcription Factors »TFIID »TFIIB »TFIIF »TFIIH

38. Transcription Factors TFIID binds to the TATA box to form the initial committed complex. TFIID has a subunit called TATA-binding protein (TBP) that recognizes the TATA box, and a number of other proteins called TBP-associated factors (TAFs). TFIID-TATA box complex acts as a binding site for TFIIB, which recruits RNA PolII and TFIIF to produce the minimal transcription initation complex. Next, TFIIE and TFIIH bind to produce the complete transcription initiation complex (or preinitiation complex, PIC).

39. Other transcription factors Activators bind to enhancers, sequences required for maximal transcription. Activator binds to an enhancer, and through an interaction with another protein called an adapter forms a bridge to the preinitation complex. In most cases, enhancers are upstream of the gene. Silencer elements have repressing ability for the transcription upon binding with a repressor.

40. Transcription by RNA Pol I Transcription of rRNA genes. rRNA genes are found in tandem repeats. A large 45S pre-RNA is transcribed, then processed to yield 28S, 18S, and 5.8S rRNAs. Promoter spans about 150 bp upstream of the initiation site. UBF (upstream binding factor) and SL1 (selectivity factor) are transcription factors that recognize the promoter. TBP is a component of SL1 and is essential. rDNA promoter does not contain TATA box.

41. rRNA gene

42. rDNA transcription

43. Transcription by RNA Pol III Synthesizes tRNA and 5S rRNA, and some small RNAs involved in splicing and protein transport. Promoter of 5S rRNA is downstream of the transcription initiation site! –TFIIIA, TFIIIC, TFIIIB, and the polymerase bind to the promoter, sequentially. tRNA promoters do not contain a binding site for TFIIIA. –TFIIIC initiates transcription. The promoter of U6 snRNA is upstream of the start site and contains a TATA box, recognized by TBP subunit of TFIIIB.

44. Transcription of polymerase III genes

45. RNA processing

46. Three of the Eukaryotic rRNAs (28S, 18S, and 5.8S rRNAs) are cleaved from a single transcript. Bacterial rRNAs (23S, 16S, and 5S) are cleaved from a single transcript. Cleavage involves several steps are somewhat different in eukaryotes and prokaryotes.

47. Processing of rRNAs

48. Processing of tRNAs tRNAs are synthesized as longer molecules, pre-tRNAs, some of which contain multiple tRNAs. The 5’ end of pre-tRNA is cleaved by an enzyme called RNaseP. RNaseP contains RNA and proteins, and RNA itself can do the cleavage. RNaseP is a ribozyme.

49. Processing of tRNAs The 3’ of tRNAs is generated by Rnase. The CCA is added into the terminus, as the site of amino acid attachment, and is required for tRNA activity during translation. Some bases (10%) are modified at characteristics places, e.g., methylguanosine, inosine, etc. They alter the base pairing properties of the tRNA molecule.

50. Processing of tRNA

52. Nonsense-mediated mRNA decay A quality control system Degredation of mRNAs that lack complete open reading frames. Takes place in cytoplasm in yeast. If a ribosome encounters a premature stop codon, this system is triggered. In mammals, it may take place in nucleus.

53. RNA degradation in cytoplasm rRNA and tRNA are stable; they are found in high levels in the cell. Bacterial mRNA have a half life of 2-3 minutes. Why? In eukaryotes, different mRNAs have different life spans (30 min to 20 hours).

54. How mRNA is degraded? Degradation is initiated by shortening of their polyA tails. 5’ cap is removed. RNA is degraded by nucleases from both ends. Which RNAs are expected to be short lived?

55. RNA degradation Shortening of poly A tails Removal of 5’ cap degradation of RNA by nucleases. IRE: Iron-response element. If iron is available, nuclease degrade the mRNA. If iron is scarce, IRE-BP binds to a sequence near 3’ protect the mRNA.