2013 W. H. Freeman and Company. 26 RNA Metabolism

Transcription

1 2013 W. H. Freeman and Company 26 RNA Metabolism

2 CHAPTER 26 RNA Metabolism Key topics: Transcription: DNA-dependent synthesis of RNA Capping and splicing: RNA processing

3 Overview of RNA Function Ribonucleic acids play three well-understood roles : Messenger RNAs encode the amino acid sequences of all the polypeptides found in the cell Transfer RNAs match specific amino acids to triplet codons in mrna during protein synthesis Ribosomal RNAs are the constituents and catalytic appropriate amino acids Ribonucleic acids play several less-understood functions: MicroRNAs regulate the expression of genes, possibly via binding to specific nucleotide sequences Ribonucleic acids act as genomic material in viruses

4 Overview of RNA Metabolism Ribonucleic acids are synthesized using DNA as a template in transcription Transcription is tightly regulated in order to control the concentration of each protein Most eukaryotic ribonucleic acids are processed after synthesis Elimination of introns; joining of exons Poly-adenylation of the 3 end Capping the 5 end

5 The footprinting technique is a way to find a DNA-binding site Premise: DNA bound by protein will be protected from chemical cleavage at its binding site. 1)Isolate a DNA fragment thought to contain a binding site 2)Radiolabel the DNA 3)Bind protein to DNA in one tube; keep another as a naked DNA control 4)Treat both samples with chemical or enzymatic agent to cleave the DNA 5)Separate the fragments by gel electrophoresis and visualize bands on X-ray film or imager plate

6 Protein-DNA Footprinting

7 Footprinting Results of RNA Polymerase Bound to Promoter

8 Transcription is a major target for regulation Transcription is energy-intensive so it s logical to regulate gene production here Regulation is achieved in many ways One way is to regulate the affinity of RNA polymerase for a promoter Promoter sequence Activator proteins Repressor proteins

9 Eukaryotes contain several distinct polymerases RNA polymerase I synthesizes pre-ribosomal RNA (precursor for 28S, 18S, and 5.8 rrnas) RNA polymerase II is responsible for synthesis of mrna Very fast ( nucleotides/sec) Specifically inhibited by mushroom toxin -amanitin Can recognize thousands of promoters RNA polymerase III makes trnas and some small RNA products Plants appear to have RNA polymerase IV that is responsible for the synthesis of small interfering RNAs Mitochondria have their own RNA polymerase

10 Features of Some Promoters Recognized by Eukaryotic RNA Polymerase II

11 Eukaryotic mrna transcription involves many proteins Relies on protein-protein contacts Many highly conserved transcription factors RNA Pol II is well-studied Large complex of 12 subunits Some subunits have some structural homology to bacterial RNA polymerase Has carboxy-terminal domain (CTD) of highly conserved repeats

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13 Transcription at RNA II Promoters

14 Assembly of RNA Polymerase at Promoter Initiated by TATA-binding protein (TBP) with the promoter -TBP is part of multisubunit complex TFIID Other proteins include TFIIB, TFIIA, TFIIF, TFIIE and TFIIH Helicase activity in TFIIH unwinds DNA at the promoter Kinase activity in TFIIH phosphorylates the polymerase at the CTD (carboxy-terminal domain) changing the conformation and enabling RNA Pol II to transcribe

15 Elongation and Termination Elongation factors bound to RNA Pol II enhance processivity and coordinate post-translational modifications For termination, Pol II is dephosphorylated

16 TFIIH and Repair Transcribed genes are more actively repaired than silent genes May partly be explained that TFIIH also has role in nucleotide-excision repair (NER) Recruits the NER complex at a lesion Genetic repair diseases are associated with TFIIH defects Xeroderma pigmentosum, etc.

17 RNA polymerases can be selectively inhibited Actinomycin D and Acridine Intercalate in DNA and prevents transcription -Amanitin from mushroom Amanita phalloides Blocks Pol II and Pol III of predators But doesn t block its own Pol II

18 Processing of mrna Overview Processing includes: Splicing out introns and rejoining any exons for a continuous sequence Adding a 5 -cap Adding a 3 -poly(a) tail Degradation

19 Maturation of mrna in Eukaryotes

20 The 5 -cap is a 7-methylguanosine 7-methylguanosine links to 5 -end via 5/,5 -triphosphate link May include additional methylations at 2 OH groups of next two nucleotides Methyl groups derive from S- adenosylmethionine (SAM) Protects RNA from nucleases Forms a binding site for ribosome

21 Introns are found in most genes Most genes in vertebrates, some in yeast, a few bacteria have introns Exons usually <1000 bp in length Introns 50 20,000 bp in length Some genes have dozens of introns

22 Introns use a 2 -OH within the intron as a nucleophile The nucleophile is a 2 -OH of an A residue within the intron After the first cleavage, the second (right-most) piece forms a lariat-like intermediate with a phosphodiester bond

23 Splicing of Intron

24 Spliceosome introns are removed via a large complex called a spliceosome Spliceosome made up of snrnps (for small nuclear ribonuclear proteins) snrnp RNA is called snrna (for small nuclear RNA) 5 snrnas known in eukaryotes (U1, U2, U4, U5, U6) GU at 5 -end and AU at 3 -end usually mark sites of splicing

25 U1 snrnp and U2 snrnp bind to the intron s ends Contain regions complementary to mrna U1 helps define the 5 -splice site U2 binds near the 3 -end of the intron Creates a bulge that partly displaces and activates an A to be a better nucleophile This A forms the 2 5 -phosphodiester bond of the lariat-like intermediate

26 Binding of U1 and U2 snrnp to mrna

27 U1 snrnp and U2 snrnp bind to the intron s ends (cont.) Next, U2, U4, U5, and U6 bind, bringing at least 50 proteins to create spliceosome ATP required for assembly but not cleavage Some parts attached to CTD (carboxyterminal domain) of RNA Pol II Indicates coordination of splicing with transcription

28 Poly(A) tail is added to eukaryotic mrnas to serve as a binding site RNA Pol II synthesizes RNA beyond the cleavage signal sequence Cleavage signal is bound by an endonuclease and a polyadenylate polymerase bound to CTD Endonuclease cleaves RNA nt downstream to highly conserved AAUAA Polyadenylate polymerase synthesizes nt of A

29 Addition of Poly(A) Tail

30 Overview of mrna Processing

31 A single gene can yield different products depending on RNA processing RNA can be edited (bases removed/added) Cleavage/polyadenylation patterns can vary, yielding different mature transcripts Immunoglobulin heavy chain gene: different degrees of polyadenylation and different cleavage sites yield diverse sequences

32 Alternative Splicing Mechanisms

33 MicroRNAs Function in Gene Regulation MicroRNAs (mirnas): Short noncoding RNAs of ~22 nucleotides Bind to specific regions of mrna to alter translation Assist in cleaving the mrnas Or block the mrna from translation ~1% of the human genome may encode mirna! Synthesized from larger precursors Processed by two endoribonucleases, Drosha and Dicer

34 Steps in mirna Processing Long precursor pri-mirna made in nucleus Drosha and DGCR8 cleave pri-mirna to a nt precursor Exportin and Ran export this precursor to the cytoplasm Dicer cleaves the pre-mirna into dsrna Complement of mirna removed by helicase mirna loaded onto protein complex such as RNAinduced silencing complex (RISC) RISC binds to target mrna

35 RISC-miRNA prevents translation of mrna (From previous slide): The mirna sequence in RISC binds to complement in target mrna If mirna is ~ perfect complement, target mrna is cleaved Thus, the mrna is not translated If mirna is only partial complement, translation is blocked

36 How mirnas are Processed to Prevent Translation

37 Cellular mrnas are degraded at different rates RNA lifetime is one means of gene regulation Half-lives vary from seconds to hours Typical vertebrate mrna ~3 hrs Shorter (~1.5 mins) half-lives for bacterial mrnas Degradation via ribonucleases Hairpin structures in mrna can extend half-life

38 Retroviruses make DNA from RNA Retroviruses have genomes of ssrna and the enzyme reverse transcriptase Virus enters host cell Reverse transcriptase makes DNA from the RNA Then degrades the RNA from the DNA-RNA hybrid and replaces it with DNA DNA can then be incorporated into host DNA

39 Retroviral Infection of a Mammalian Cell and Integration into Host Chromosome

40 Retroviruses typically contain three genes plus a long terminal repeat gag (group associated antigen) pol env Encodes a long polypeptide that is cleaved into six smaller proteins that make up viral core Encodes protease that cleaves the long polypeptide, reverse transcriptase, and an integrase to insert DNA into host genome Encodes viral envelope Long terminal repeat (LTR) facilitates integration of virus genome into host DNA

41 Structure and Gene Products of an Integrated Retroviral Genome

42 Reverse transcriptases catalyze three reactions 1) RNA-dependent DNA synthesis 2) RNA degradation 3) DNA-dependent DNA synthesis Contain Zn 2+, like DNA Pol Use a primer of trna Lack 3 5 -proofreading, like RNA Pol - Make reverse transcriptase error-prone - Explains high rate of virus mutation/evolution

43 Some retroviruses cause cancer Some retroviruses contain an oncogene. Example: Rous sarcoma virus has the src gene Src for sarcoma, a cancer of bone, fat, muscle, etc. Encodes a non-receptor tyrosine kinase, an enzyme that affects cell division Rous Sarcoma Virus Genome

44 HIV retrovirus causes AIDS HIV genome has genes for killing host (mostly T lymphocytes) Results in suppression of immune system HIV-encoded reverse transcriptase is unusually error-prone Complicates push for vaccine At least one error per replication, so potentially no two viral RNAs alike

45 HIV Genome

46 Pharmaceutical Targets for HIV (Antiretroviral Drugs) Reverse transcriptase inhibitors Nucleotide or nucleoside analogs Zidovudine (AZT), Didanosine (Videx), etc. Protease inhibitors Since proteases used in cleaving proteins for packaging into new viral particles Indinavir, Saquinavir, etc.

47 Retrotransposons in eukaryotes have similarities to retroviruses Retrotransposons are mobile genetic elements in eukaryotes Encode an enzyme with homology to reverse transcriptase of retroviruses Move between positions via RNA intermediates Using their enzyme to make DNA from RNA unlike bacterial transposons that move directly from DNA to DNA Lack the env gene so don t form viral particles

48 Eukaryotic Transposons

49 Telomeres Structures at the ends of eukaryotic chromosomes Have tandem repeats usually of T 1-4 G With A-C on the opposing strand Can be tens of thousands of bp long in mammals TG strand is longer than its complement, leaves a 3 -overhang of several hundred bases

50 Telomerase extends the ends of linear chromosomes Telomeres are not easily replicated using DNA polymerases Beyond an end there is no template for an RNA primer Chromosomes are shortened with each generation Telomerase adds telomeric sequences to solve this problem

51 The Mechanism of Telomerase Telomerase has RNA with C y A x repeat to serve as template for synthesis of the T x G y strand of the telomere Telomerase extends the 3 -end, using the RNA of the enzyme as the primer The gap on the bottom strand is filled in by DNA polymerases

52 Telomerase Mechanism

53 Chapter 26: Summary In this chapter, we learned: RNA polymerase synthesizes RNA using a strand of DNA as a template and nucleoside triphosphates as substrates The primary RNA transcript in eukaryotes requires processing before it becomes messenger RNA The processing involves capping 5 end with methylguanosine to stabilize the RNA molecule The processing involves splicing out introns