Transcription of Protein-Coding Genes and Formation of Functional mRNA

1. Transcription of Protein-Coding Genes and Formation of • Functional mRNA • Gene= “unit of DNA---information to specify synthesis of a single polypeptide chain or functional RNA e.g tRNA).” • protein-coding genes = mRNA molecules of cells. • DNA of viruses= a few genes • single DNA in each of chromosomes of higher animals & plants= several thousand genes.

2. During Transcription 4 bases transcribed? • During protein synthesis 4 base language of DNA and RNA--translated into20–amino acid language of proteins. • formation of functional mRNAs from protein-coding genes • similar to precursors of rRNAs and tRNAs encoded by rRNA & • tRNA genes----further modified to functional rRNAs and tRNAs

3. A Template DNA Strand Is Transcribed into a Complementary RNA Chain by RNA Polymerase • One DNA strand acts as a template, determining order in which (rNTPs) monomers polymerized to form a complementary RNA chain • DNA base-pair with complementary incoming rNTPs--- joined in a polymerization reaction catalyzed by RNA polymerase. • Polymerization: a nucleophilic attack by 3_ oxygen in growing RNA chain on _phosphate of next nucleotide precursor to be added---- phosphodiester bond & release of pyrophosphate (PPi) • -RNA molecules synthesized in 5_n3_ direction

4. Polymerization reaction : addition of rNTPs to growing RNA– high • energy bond between α &β phosphate of rNTP monomers replaced by lower-energy phosphodiester bond • Equilibrium for reaction driven toward chain elongation by pyrophosphatase=catalyzes cleavage of PPi into2 inosrganic phosphate • -template DNA strand and growing RNA strand :bp to it have opposite • 5_n3_ directionality • -Site at which RNA polymerase begins transcription: +1.

5. -Downstream= direction in which a template DNA strand transcribed/mRNA translated --downstream sequence toward 3’ end relative to start site -Nucleotide positions in DNA downstream from a start site indicated a positive +sign -Upstream= opposite direction ; those upstream, by a negative - sign.

6. Stages in Transcription • RNA polymeras functions: • 1-initiation,RNA polymerase recognizes & binds to promoter • RNA polymerases require protein factors: general transcription factors: locate promoters and initiate transcription • 2- RNA polymerase melts DNA strands ( 14bps around start site): bases in template available for base pairing with bases Of rNTPs • 3-Transcription initiation complete when first two rNTPS of an RNA chain linked by bond)

7. 4-After several rNTPs polymerized, RNA polymerase dissociates from promoter DNA & general transcription factors. 5- strand elongation, RNA polymerase moves along template DNA one base at a time, opening ds DNA in front of movement and hybridizing strands behind it ---enzyme maintains a melted region of 14 bp=transcription bubble. ----8 nucleotides at 3_ end of growing RNA strand remain bp to template DNA strand in transcription bubble.

8. -Elongation complex(stable): RNA polymerase, template DNA & growing (nascent) RNA strand, • -RNA polymerase transcribes longest mammalian genes without dissociating from DNA template or releasing nascent RNA. • RNA synthesis occurs at a rate of 1000 nt/ min at 37C • ---elongation complex remain intact more24 hrs to assure continuous RNA synthesis

9. -Transcription termination : final stage in RNA synthesis: primary transcript E D I A C O N N E C T I O N S • released from RNA polymerase----polymerase dissociates from template DNA • -Specific sequences in template DNA signal the bound RNA polymerase to terminate transcription • Once released RNA polymerase free to transcribe the same gene again or another gene.

10. Structure of RNA Polymerases • RNA polymerases of bacteria, archaea, & eukaryotic cells similar in structure and function • Bacterial RNA polymerases: 2 large subunits β and β’, 2 subunits α & one copy • of ω : not essential for transcription or cell viability but stabilizes enzyme & assists in assembly of its subunits. • -Archaeal & eukaryotic RNA polymerases : additional small subunits associated with this core Complex , will be discussed later(e.g Ϭ: sigma) • -RNA polymerase bound to unbent DNA, but DNA bends according to interaction between bacterial RNA polymerase & promoter DNA

11. Organization of Genes Differs in Prokaryotic and Eukaryotic DNA • Sequencing of genomes: revealed variations in number of protein-coding genes • & differences in organization in prokaryotes and eukaryotes. • -Common arrangement of protein-coding genes in prokaryotes: • Operon: operates as a unit from a single promoter • Transcription of an operon produces a continuous strand of mRNA--carries message for a related series of proteins • - Each section of mRNA represents gene encodes one of proteins in series. • - In prokaryotic DNA genes packed with very few noncoding gaps, DNA • transcribed directly into colinear mRNA----translated into protein.

12. -Clustering of genes devoted to a single metabolic function does not occur in eukaryotes & yeasts -eukaryotic genes devoted to a single pathway, physically separated in DNA--located on different chromosomes -Each gene transcribed from its own promoter—one mRNA-- translated to single polypeptide Exons, introns(euk. And virus: common, bacteria:rare, yeast:lack)

13. Eukaryotic Precursor mRNAs Processed to Form Functional mRNAs • Prokaryotic cells, translation of mRNA begin from the 5_ end even 3_ end still by RNA polymerase. • - transcription and translation occur concurrently? • -Eukaryotic cells: primary transcripts of protein-coding genes : precursor mRNAs • (pre-mRNAs) • --------- RNA processing--- functional mRNA • - mRNA then exported to cytoplasm • transcription and translation cannot occur concurrently. • -

14. All eukaryotic pre-mRNAs modified at two ends 5’ , 3’? Cap—function?? • -protects an mRNA from degradation, assists export to & bound by a protein factor required to begin translation in cytoplasm • Processing at the 3_ end of a pre-mRNA ?poly(A) polymerase (no template needed).---poly(A) tail . • - Final step in processing of mRNA= RNA splicing? • .

15. -Functional eukaryotic mRNAs produced by RNA • processing retain noncoding regions= 5’&3’UTRs, • In mammalian mRNAs, 5’ UTR shorter than 3’UTR • Prokaryotic mRNAs have 5’& 3’ UTRs, but shorter than in eukaryotic mRNAs

16. Control of Gene Expression in Prokaryotes • Control of gene expression fundamental aspect of molecular cell biology • -Controlling transcription initiation--- regulate which proteins it produces and how rapidly. • transcription repressed-- mRNA and protein or proteins synthesized at low rates. • -transcription of a gene activated mRNA and encoded proteins produced at higher rates. • -In bacteria & single-celled organisms—gene expression regulated---to adjust cell’s enzymatic machinery & structural components to changes in nutritional and physical environment. • -any given time, a bacterial cell synthesizes proteins required for survival under particular • conditions.

17. WHILE -In multicellular , control of gene expression directed toward assuring that right gene expressed in right cell at right time during embryological development & tissue differentiation.

18. e.g lac operon in E. Coli: encodes 3 enzymes involved in required for the metabolism of lactose e.g trp operon encodes 5 enzymes needed in biosynthesis of tryptophan 4.3 -Transcription of operons controlled by an interplay between RNA polymerase & repressor & activator proteins. -To initiate transcription E. coli RNA polymerase associated with sigma factors e.g Ϭ 70= initiation factors.

19. Initiation of lac Operon Transcription Can Be Repressed &Activated • -When E. coli in environment lacks lactose—synthesis of lac mRNA repressed-- cellular energy not wasted synthesizing enzymes • In environment containing both lactose & glucose-- metabolize glucose--- • -Lactose metabolized at a high rate only when lactose present and glucose depleted from the medium. • -Transcription of lac operon under different conditions controlled by lac repressor binds Operator & catabolite activator protein (CAP)--binds to a specific DNA sequence in lac transcription-

20. -operator overlaps transcription start site--blocks transcription initiation by polymerase • When lactose present---- binds to specific binding sites in each subunit of tetrameric lac repressor-------conformational change in protein makes it dissociate from lac operator. • ------ polymerase initiate transcription of lac operon. • -When glucose present: rate of transcription initiation: number of times/ min different polymerase initiate transcription very low---- synthesis of low levels of lac mRNA & proteins

21. -Once glucose depleted media & intracellular glucose concentration falls— -cells respond by synthesizing cyclic AMP--- binds to a site in each subunit of the dimeric CAP protein--- conformational change ---- protein bind to CAP site in lac transcription-control region. -Bound CAP-cAMP complex interacts with polymerase bound to promoter--- stimulating rate of transcription initiation. ------activation leads to synthesis of high levels of lac mRNA & enzymes encoded

22. -Promoters for different E. coli genes exhibit homology--- exact sequences differ. • promoter sequence determines intrinsic rate at which an RNA • polymerase– complex initiates transcription of a gene in absence of a repressor or activator protein. • -Promoters support a high rate of transcription initiation=strong promoters. • -Promoters support a low rate of transcription initiation= weak promoters. • - lac operon has a weak promoter: low intrinsic rate of initiation reduced by lac repressor & increased by cAMP-CAP activator

23. Small Molecules Regulate Expression of Many Bacterial Genes via DNA-Binding Repressors A small molecule=inducer binds to repressor--controlling its DNA-binding activity e.g tryptophan concentration in medium & cytosol high--- cell does not synthesize enzymes -Binding of tryptophan to trp repressor----conformational change--- protein to bind to the trp operator, ----repressing expression of enzymes synthesize tryptophan.

24. when tryptophan concentration in medium & cytosol low--- tryptophan dissociates from trp repressor-- a conformational change in protein ---- dissociate from the trp operator---transcription of the trp operon. • lac operon, binding of inducer lactose to lac repressor reduces binding of repressor to operator---transcription. • -Activator proteins e.g CAP in lac operon, control transcription of some but not all bacterial genes. • -activators bind to DNA & RNA polymerase----stimulating transcription

25. Transcription by Ϭ 54-RNA Polymerase Controlled by Activators That Bind Far from Promoter - E. coli promoters interact with Ϭ 70-RNA polymerase(several alternative Ϭ factors recognize different consensus promoter sequences. -Sequence of one E. coli sigma factor, Ϭ 54, different from that of Ϭ 70-like factors. -Activators binding sites = enhancers( 80–160 b p upstream from start site

26. -Best-characterized Ϭ 54-activator—NtrC protein (nitrogen regulatory protein C)—stimulates transcription from promoter of the glnA gene. • - gene encodes glutamine synthetase( synthesizes amino acid glutamine from glutamic acid & ammonia) • Ϭ 54-RNA polymerase binds to glnA promoter but does not melt DNA strands & initiate transcription until it is activated by NtrC, a dimeric protein. • NtrC, regulated by a protein kinase called NtrB.

27. Regulatory Elements in Eukaryotic DNA :Kilobases from Start Sites RNA polymerase binds promoter. controlled by DNA-binding proteins =transcription factors(TF)= bacterial repressors &activators. DNA control elements in eukaryotic genomes bind TF located farther (Upstream: opposite to transcription /downstream: same direction of transcription)from promoter than in prokaryotic genomes.

28. Three Eukaryotic Polymerases Catalyze Formation of Different RNAs 3 RNA polymerases: I, II, & III. -Eluted at different salt concentrations during ion-exchange chromatography & differ in sensitivity: To amanitin( poisonous cyclic octapeptide by mushrooms) Polymerase I :insensitive, polymerase II very sensitive; polymerase III intermediate sensitivity.

29. RNA polymerase I, located in nucleolus: transcribes genes encoding precursor rRNA (pre-rRNA) • -processed into 28S, 5.8 & 18S rRNAs. • RNA polymerase III transcribes genes encoding tRNAs, 5S rRNA, & an array of small, stable RNAs, including one involved in RNA splicing (U6) & RNA component of signal-recognition particle (SRP) involved in directing nascent proteins to ER • RNA polymerase II transcribes all protein-coding genes( mRNAs) & produces four of five small nuclear RNAs that take part in RNA splicing.

30. Best characterized eukaryotic RNA polymerases from yeast S. cerevisiaes 3 eukaryotic RNA polymerases more complex than E. coli RNA polymerase BUT structures Similar - All three contain two large subunits (Similar to β & β’s E.coli Subunits) & 10–14 smaller subunits - eukaryotic polymerases also contains ω-like & two nonidentical α-like subunits.

31. The Largest Subunit in RNA Polymerase II Has Essential Carboxyl-Terminal Repeat • Carboxyl end of largest subunit of RNA polymerase II,ONLY, (RPB1) contains a stretch of 7 amino acids,repeated multiple times= heptapeptide = terminal domain (CTD). • CTD critical for viability • -In vitro experiments with model promoters first showed RNA polymerase II initiate transcription have an unphosphorylated CTD • Once polymerase initiates transcription & begins to move away from the promoter many of the serine and some tyrosine residues in CTD phosphorylated.

32. Regulatory Sequences in Protein-Coding Genes expression of eukaryotic protein-coding genes regulated by multiple protein-binding DNA sequences= transcription control regions. - promoters and other elements located near transcription start sites & sequences located far from genes they regulate. Properties of control elements:. The TATA Box, Initiators, and CpG Islands ,Function as Promoters in Eukaryotic DNA

33. 1- Aconserved sequence=TATA box -25-35 bp upstream of start site - TATA box acts similarly to an E. Coli promoter to position RNA polymerase II for transcription 2-an alternative promoter element=initiator - Directed mutagenesis experiment:---- nucleotide sequence immediately surrounding start site determines strength of promoters.

34. -Transcription of genes with promoters containing a TATA box or initiator element begins at a well-defined initiation site. -“housekeeping genes” do not contain a TATA box or an initiator transcribed at low rates (e.g., genes encoding enzymes of intermediary metabolism - contain a CG-rich stretch of 20–50 nucleotides TATA-box or initiator sequences that determine initiation site in template=promoter-proximal elements 3- Dinucleotide CG =CpG island, just upstream from a start site ,suggests may contain a transcription-initiation region.

35. 4- control elements located thousands of base pairs away from =enhancers, • common in eukaryotic genomes but fairly rare in bacterial genomes. • enhancers and promoter-proximal elements : • - both types of element stimulate transcription even when inverted • -both cell-type-specific

36. Activators and Repressors of Transcription Activators Modular Proteins Composed of Distinct Functional Domains -functional domains: N-terminal DNA-binding domain, C-terminal activation domain, which interacts with other proteins to stimulate transcription from a nearby promoter Repressors Functional Converse of Activators - constitutive expression=high expression (on)---inactivation of a repressor -have two functional domains:a DNA-binding domain and a repression domain.

37. DNA-Binding Domains Can Be Classified into Numerous Structural Types • DNA-binding domains of eukaryotic activators and repressors contain structural motifs: bind specific DNA sequences. • ability of DNA-binding proteins to bind to DNA sequences----noncovalent interactions between atoms in an _ helix in DNA-binding domain and atoms on the edges of bases within a major groove in DNA. • -Interactions with sugar phosphate backbone atoms and, in some cases, with atoms in a DNA minor groove

38. General Transcription Factors Position RNA Polymerases II at Start Sites and Assist in Initiation - general transcription factors = initiation factors (from TATA box), position polymerase molecules at transcription start sites and help to melt DNA strands -------------------strand can enter active site of enzyme -e.g TFIIA, TFIIB, etc.(multimeric 0proteins) -largest is TFIID: TATA box–binding protein (TBP) and TBP associated factors (TAFs) - general transcription factors from different eukaryotes highly conserved.

39. Sequential Assembly of Proteins Forms the Pol II Transcription Preinitiation Complex in Vitro • Pol II preinitiation complex= Pol II molecule and general transcription factors bound to a promoter region of DNA • TBP first protein to bind to a TATAbox promoter. • - All eukaryotic TBPs analyzed have similar C-terminal domains • -The N-terminal domain of TBP, varies in sequence and length among different eukaryotes • - functions in Pol II–catalyzed transcription of genes encoding snRNAs

40. Molecular Mechanisms of Transcription Activation and Repression -activators and repressors that bind to specific sites in DNA and regulate expression by two mechanisms 1-regulatory proteins act in concert with other proteins to modulate chromatin structure, thereby influencing the ability of general transcription factors to bind to promoters. -DNA in eukaryotic cells associated protein= chromatin. - basic structural unit of chromatin= nucleosome= DNA wrapped tightly around a disk-shaped core of histone proteins.

41. -Residues within the N-terminal region of each histone, and the C-terminal region of histone H2A, =histone tails, extend from surface of the nucleosome, -modifications=acetylation of histone H3 and H4 tails, influence the relative condensation of chromatin & its accessibility to proteins required for transcription initiation. -activators & repressors interact with a large multiprotein complex called = mediator .-------binds to Pol II and directly regulates assembly of transcription preinitiation complexes.

42. Repressors Can Direct Histone Deacetylation at Specific Genes deacetylation of histone tails in nucleosomes that bind to TATA box and promoter-proximal region of genes they repress - In unacetylated histones, N-terminal lysines positively charged and interact strongly with DNA phosphates. -unacetylated histone tails interact with neighboring histone octamers, favoring folding of chromatin into condensed---- general transcription factors cannot assemble into a preinitiation complex

43. -In contrast, binding of general transcription factors repressed much less by histones with hyperacetylated tails ------- positively charged lysines neutralized and electrostatic interactions with DNA phosphatese eliminated.

44. Transcription of Many Genes Requires Ordered Binding of Activators and Action of Co-Activators Accessory proteins =Co-activators ---function to make genes within nucleosomal DNA accessible to general transcription factors & Pol II & directly recruit Pol II to promoter regions.