Chapter 11 Transcription and RNA Processing

1. Chapter 11Transcription and RNA Processing José A. Cardé Serrano, PhD Universidad Adventista de las Antillas Biol 223 – Genética Agosto 2010

2. Chapter Outline - Objetivos • Transfer of Genetic Information: The Central Dogma • The Process of Gene Expression • Transcription in Prokaryotes • Transcription and RNA Processing in Eukaryotes • Interrupted Genes in Eukaryotes: Exons and Introns • Removal of Intron Sequences by RNA Splicing

3. The central dogma of biology is that information stored in DNA is transferred to RNA molecules during transcription and to proteins during translation. Transfer of Genetic Information:The Central Dogma

4. The Central Dogma

5. Transcription and Translation in Prokaryotes • The primary transcript is equivalent to the mRNA molecule. • The mRNA codons on the mRNA are translated into an amino acid sequence by the ribosomes.

6. Transcription and Translation in Eukaryotes • The primary transcript (pre-mRNA) is a precursor to the mRNA. • The pre-mRNA is modified at both ends, and introns are removed to produce the mRNA. • After processing, the mRNA is exported to the cytoplasm for translation by ribosomes.

7. Types of RNA Molecules • Messenger RNAs (mRNAs)—intermediates that carry genetic information from DNA to the ribosomes. • Transfer RNAs (tRNAs)—adaptors between amino acids and the codons in mRNA. • Ribosomal RNAs (rRNAs)—structural and catalytic components of ribosomes. • Small nuclear RNAs (snRNAs)—structural components of spliceosomes. • Micro RNAs (miRNAs)—short single-stranded RNAs that block expression of complementary mRNAs.

9. Key Points • The central dogma of molecular biology is that genetic information flows from DNA to DNA during chromosome replication, from DNA to RNA during transcription, and from RNA to protein during translation.

10. Key Points • Transcription involves the synthesis of an RNA transcript complementary to one strand of DNA of a gene. • Translation is the conversion of information stored in the sequence of nucleotides in the RNA transcript into the sequence of amino acids in the polypeptide gene product, according to the specifications of the genetic code.

11. Information stored in the nucleotide sequences of genes is translated into the amino acid sequences of proteins through unstable intermediaries called messenger RNAs. The Process of Gene Expression

12. RNA Synthesis And Transport in Eukaryotes • Method: Pulse-Chase Labeling • At first, labeled RNA is exclusively in the nucleus. • Later, the labeled RNA is found in the cytoplasm. • RNA is synthesized in the nucleus and then transported to the cytoplasm.

13. General Features of RNA Synthesis • Similar to DNA Synthesis except • The precursors are ribonucleoside triphosphates. • Only one strand of DNA is used as a template. • RNA chains can be initiated de novo (no primer required). • The RNA molecule will be complementary to the DNA template (antisense) strand and identical (except that uridine replaces thymidine) to the DNA nontemplate (sense) strand. • RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5’3’ direction.

16. The Transcription Bubble

17. Key Points • In eukaryotes, genes are present in the nucleus, whereas polypeptides are synthesized in the cytoplasm. • Messenger RNA molecules function as intermediaries that carry genetic information from DNA to the ribosomes, where proteins are synthesized.

18. Key Points • RNA synthesis, catalyzed by RNA polymerases, is similar to DNA synthesis in many respects. • RNA synthesis occurs within a localized region of strand separation, and only one strand of DNA functions as a template for RNA synthesis.

19. Transcription—the first step in gene expression—transfers the genetic information stored in DNA—genes—into messenger RNA molecules that carry the information to the ribosomes—the sites of protein synthesis—in the cytoplasm. Transcription in Prokaryotes

20. Stages of Transcription

21. E. Coli RNA Polymerase • Tetrameric core: 2’ • Holoenzyme: 2’  • Functions of the subunits: • : assembly of the tetrameric core • : ribonucleoside triphosphate binding site • ’: DNA template binding region • : initiation of transcription

22. Initiation of RNA Chains • Binding of RNA polymerase holoenzyme to a promoter region in DNA • Localized unwinding of the two strands of DNA by RNA polymerase to provide a single-stranded template • Formation of phosphodiester bonds between the first few ribonucleotides in the anscent RNA chain

23. Numbering of a Transcription Unit • The transcript initiation site is +1. • Bases preceding the initiation site are given minus (–) prefixes and are referred to as upstream sequences. • Bases following the initiation site are given plus (+) prefixes and are referred to as downstream sequences.

24. A Typical E. coli Promoter • Consensus sequences: -10 sequence and -35 sequence • Recognition sequence: -35 sequence

25. Elongation

26. Termination Signals in E. coli • Rho-dependent terminators—require a protein factor () • Rho-independent terminators—do not require 

27. Rho-Independent Termination

28. Coupled Transcription and Translation in E. coli

29. Key Points • RNA synthesis occurs in three stages: (1) initiation, (2) elongation, and (3) termination. • RNA polymerases—the enzymes that catalyze transcription—are complex multimeric proteins. • The covalent extension of RNA chains occurs within locally unwound segments of DNA.

30. Key Points • Chain elongation stops when RNA polymerase encounters a transcription-termination signal. • Transcription, translocation, and degradation of mRNA molecules often occur simultaneously in prokaryotes.

31. Three different enzymes catalyze transcription in eukaryotes, and the resulting RNA transcripts undergo three important modifications, including the excision of noncoding sequences called introns. The nucleotide sequenced of some RNA transcripts are modified posttranscriptionally by RNA editing. Transcription and RNA Processing in Eukaryotes

33. Modifications to Eukaryotic pre-mRNAs • A 7-Methyl guanosine cap is added to the 5’ end of the primary transcript by a 5’-5’ phosphate linkage. • A poly(A) tail (a 20-200 nucleotide polyadenosine tract) is added to the 3’ end of the transcript. The 3’ end is generated by cleavage rather than by termination. • When present, intron sequences are spliced out of the transcript.

34. Eukaryotes Have Three RNA Polymerases

35. A Typical RNA Polymerase II Promoter

36. Initiation by RNA Polymerase II TFIID se une a TATA box TFIID – contiene TBP TFIIA se une al complejo TfIIB Dnase Fingerprinting

37. Initiation by RNA Polymerase II TFIIF – helicasa - iniciacion - se asocia primero a la pol y luego ambos al complejo Polimerasa II TFIIE TFIIH y TFIIJ -ubicacion desconocida H – helicasa para extension

38. Structure of Yeast RNA Polymerase II - Observar los zurcos donde se enlaza el DNA y donde se libera el RNA nasciente

39. The 7-Methyl Guanosine (7-MG) Cap • 1ra modificacion del preMRNA • adicion de guanosinsa metilada • enlace 5’-5’ • se anade cotranscripcional • 2 funciones • iniciacion de traduccion • estabilidad/proteccion

40. The 3’ Poly(A) Tail • Terminación - • -por corte endonucleolitico del pre mRNA • varios puntos, 1000-2000 down de donde sera el 3’ • AAUAA y GU • poliA pol añade 200 A • 2 funciones: • estabilidad • transporte

41. RNA Editing • Usually the genetic information is not altered in the mRNA intermediary. • Sometimes RNA editing changes the information content of genes by • Changing the structures of individual bases • Inserting or deleting uridine monophosphate residues.

42. Editing of Apoplipoprotein-B mRNA Edicion de modificar CU 4563 aa vs 2153 aa UAA – Codon de terminacion prematuro Deaminacion de C Insercion de bases usando los RNA guias mitocondrias y Tripanosomas

43. Key Points • Three different RNA polymerases are present in eukaryotes, and each polymerase transcribes a distinct set of genes. • Eukaryotic gene transcripts usually undergo three major modifications: • the addition of 7-methyl guanosine caps to t’ termini, • The addition of poly(A) tails to 3’ ends, and • The excision of noncoding intron sequences.

44. Key Points • The information content of some eukaryotic transcripts is altered by RNA editing, which changes the nucleotide sequences of transcripts prior to their translation.

45. Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, or exons. The introns are excised from the RNA transcripts prior to their transport to the cytoplasm. Interrupted Genes in Eukaryotes: Exons and Introns

46. The Discovery of Introns

47. Introns • Introns (or intervening sequences) are noncoding sequences located between coding sequences. • Introns are removed from the pre-mRNA and are not present in the mRNA. • Exons (both coding and noncoding sequences) are composed of the sequences that remain in the mature mRNA after splicing. • Introns are variable in size and may be very large.

48. Key Points • Most, but not all, eukaryotic genes are split into coding sequences called exons and noncoding sequences called introns. • Some genes contain very large introns; others harbor large number of small introns. • The biological significance of introns is still open to debate.

49. The noncoding introns are excised from gene transcripts by several different mechanisms. Removal of Intron Sequences by RNA Splicing

50. Excision of Intron Sequences