Chapter 11 Transcription and RNA Processing José A. Cardé Serrano, PhD Universidad Adventista de las Antillas Biol 223 – Genética Agosto 2010 El mRNA es como un diamante sin pulir, explique: Computadoras usan codigo binarios de 0 y 1 Clave morse usa codigo de puntos y rayas, dos simbolos Alfabeto … 26 letras Como la info genetica en organismos vivos es escrita en un alfabeto de solo 4 letras o bases Como la informacion genetica es expresada durante el desarrollo del organismo el RNA tiene un rol protagonico

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

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

The Central Dogma

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.

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.

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.

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.

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.

The Process of Gene Expression Information stored in the nucleotide sequences of genes is translated into the amino acid sequences of proteins through unstable intermediaries called messenger RNAs. Genes estan enel nucleo, proteinas se fabrican en el cito entonces Como estos genes controlan el proceso lejos de ellos? Hipoteisis : una molecula intermediaria q lleva la informacion de unl ugar a otro..

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. las primeras evidencias se dieron en procariotes Evidencias de un itermediario de RNA por tecncas de radioisotopos: pulse autoradiografica Pulso con 3H Uridina y se detiene – la radioactividad se ve en el nucleo solamente Pulso y seguimiento con uridina fria – la radioatvidad se ve en el citoplasma PLT el mRNA se fabrica en el nucleo y se usa en el cito

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.

Diferencias Enfasis en Sense y nonsense 0 non template y template:

Direccion de la sintesis de RNA ocurre 53

The Transcription Bubble Promotores – secuencias especificas donde se pega la po.imerasa junto a facts de transcripcion inciain el proceso cerca e los promotores \ En procariotes hay 1 sola RNA pol en Eucariotes hay 3 RNA pol para distintos tipos de RNA Lugar dondde se realiza la sintesis de RNA. BURBUJA DE transcripcion

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.

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.

Transcription in Prokaryotes 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.

Stages of Transcription Transcripcion es basicamente igual en procariotes y eucariotes pero tambien hay diferencias Unidad transcripcional – un segmento de DNA que es transcrito pueden ser genes individuales en procariotes pueden haber varios genes en una sola secuencia Los tres pasos de la transcripcion son: - iniciacion- alargamiento terminacion upstream y downstream – regiones localizadas hacia el 5’ o hacia el 3’ respectivamente

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 RNA pol – enzimas multimericas la de E coli tiene 5 polipeptidos… 2 son iguales por lo tanto tienen 4 polipeptidos Holoenzima es la enzima completa, cada sub U tiene una funcion especifica Sigma es para binding e iniciacion yluego se sale y sigue el core extendiendo

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 Iniciacion tiene 3 pasos: Sigma se despega luego delos primeros 8 – 10 nucleotidos nuevos Se usan signos + o - para referirse a zonas antes y despues del lugar de iniciacion

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.

A Typical E. coli Promoter promotores tienen secuencias en comun o consensos y conservadas varian de gen en gen pero algunas bases son conservadas consenso las conservadas en la -10 las bases son TATAAT en la - 35 las bases son TTGACA Sigma reconoce y se pega a la -35 las -10 es rica en TA Consensus sequences: -10 sequence and -35 sequence Recognition sequence: -35 sequence

Elongation 2do paso es alargamiento – Catalizada por el core de la RNA pol (sin sigma) La RNA Pol tiene actividad de enrollar y desenrollar abre un area como de 18 ntd y anade 40 nct por seg solo pareo de 3 bases en el hibrido y la estabilidda se mantiene porq ambas cadenas estan unidas a la RNA POl

Termination Signals in E. coli Rho-dependent terminators—require a protein factor () Rho-independent terminators—do not require  Para terminacion hay una senal que la RNA pol encuentra y reconoce al llegar ahi el complejo se disocia suelta la cadena naciente rho - u

Rho-Independent Termination

Coupled Transcription and Translation in E. coli En procario los dos procesos son acoplados transcripcion y traduccion y degradacion al mismo tiempo sobre una misma hebra de DNA los tres procesos ocurren 53 En procariotes no hay compartamentalizacion

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.

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.

Transcription and RNA Processing in Eukaryotes 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. Proceso mucho mas complejo en eucariotes Compartamentalizacion – Sintesis en el nucleo traducion en el citoplasma por lo general mRNA multigenicos en procariotes vs monogenicos en eucariotes Excepction C elengans tiene multigenicos 1 RNA pol vs 3 RNA POL EN EUCARIOTAS MODIFICACIONES EN EUCARIOTAS VS PROCARIOTAS

MODIFICACIONES EN EL MRNA EUCARIOTA 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.

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. El CAP 5’ ES UNT METILGUANOSINA ENLACE 5 – 5 POBLACION E MRNAS ES CONOCIDA COMO HETEROGENEO NUCLEAR RNA (hnRNA) mucha variedad de tamanos esa variacion en tamano es dad por intrones – secuencias q no codifican para proteinas lso transcriptos primarios o pre-mRNA cubiertos de proteinas q ayudan a estabilidad media vida 5 horas vs 5 min en procariotas

Eukaryotes Have Three RNA Polymerases 3 polimerasas para 3 sets de genes Requieren factorres de transcripcion distinto al a de procariotas Distintas sensibilidades a alfa amanitina RNA pol I insensible al inhibidor Pol II inhibida completamente por bajas [ ] pol III inhibicion intermedia Usarla para determinar cual pol es responsible de la transcripcion de cual gen No pueden iniciar la reaccion por si solas requireesn factores

A Typical RNA Polymerase II Promoter Una RNA pol II tipica – una RNA pol dependiente de DNA transcribe genes estructirales desenredar la doble helice con la ayuda de TF q se pegan a secuencias especificas promotor visto aqui distintas secuencias TATAAAA y GGCCAATCT elementos de secuencias conservados -30 y -80 GC box Reconocer iniciar efeiciencia

Initiation by RNA Polymerase II TFIID se une a TATA box TFIID – contiene TBP TFIIA se une al complejo TfIIB Dnase Fingerprinting Factores basales de transcripcion para iniciar enhancer y silencer para modular la eficiencia TF en un orden especifico TFII x – letras y orden de funcion no van…letra va con orden de descubrimiento Donde se pega cada factor enel DNA se determina con exptos de proteccion de degradacion en I termedios Dnase fingerprinting oligos con la secuecia putatitva marcados radioactivamente en un extremo incubar la mitad con la proteina de interes o el TF y la otra no Tratarlo scon DNAsa pancreatica I. brevemente para solo un corte (hace cortes al azar en el dna) Electroforesis la muestra sin proteinas tendra pedazos de todos los tamanos posibles La muestra con las proteinas tendra pedazos similares y algun pedazo faltara o sera mas gde la proteina no dejo q lo cortaran PLT esa secuencia fue protegina

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 Las RNA pol I y II realizan procesos similares con pequenas diferencias mas simples diferencias en los promotores - I – promotores bipartitas divididos en dos upstream III – promotores dentro de la unidad transcripcional, downstream del lugar de inicio, y algunos ubpstream como los de la II

Structure of Yeast RNA Polymerase II - Observar los zurcos donde se enlaza el DNA y donde se libera el RNA nasciente Una vez la polimerasa se suelta del complejo de iniciacion, sigue con el alargamiento Por un lado se une al DNA templado y por otro lado sale la molecula nueva

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 Temprano en el proceso cuando van solo 30 nct

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 FACT – para evadir nucleosomas convierte octamero en hexameros remuven H2A/H2B histonas acetiladas tambien facilintan

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.

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 Ejemplo de Editing de mRNA o del intermediario

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.

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.

Interrupted Genes in Eukaryotes: Exons and Introns 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. En procariotas los genes son continuos y hay colinearidad entre el Gen y el mRNA y el polipeptido En eucariota el gen de B globina se encontro q contenia regiones q no eran traducidas en proteinas Intrones Exones La mayoria de los genes tienen intrones EJ – VTG de Xenopus – tiene 33 intrones Colageno de ave tiene 50 intrones – resultado: ungen de 37,000 da un mRNA de 4600 nct

The Discovery of Introns Heteroduplex de mRNA y DNA del genoma de Adenovirus

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.

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.

Removal of Intron Sequences by RNA Splicing The noncoding introns are excised from gene transcripts by several different mechanisms. El proceso de remover intrones debe ser bien preciso a nivel de no fallar ni por una base Unir dos exones para mantener el marco de lectura Requerira senales precisas q controlen el proceso Senales son secuencias en el intron y en el exon y en los puntos de union

Excision of Intron Sequences

Splicing Removal of introns must be very precise. Conserved sequences for removal of the introns of nuclear mRNA genes are minimal. Dinucleotide sequences at the 5’ and 3’ ends of introns. exon-GT…………AG-exon TACTAAC box about 30 nucleotides upstream from the 3’ splice site.

Types of Intron Excision The introns of tRNA precursors are excised by precise endonucleolytic cleavage and ligation reactions catalyzed by special splicing endonuclease and ligase activities. The introns of some rRNA precursors are removed autocatalytically in a unique reaction mediated by the RNA molecule itself. The introns of nuclear pre-mRNA (hnRNA) transcripts are spliced out in two-step reactions carried out by spliceosomes.

The Spliceosome RNA/protein structure Excises introns from nuclear pre-mRNA Five snRNAs: U1, U2, U4, U5, and U6 Some snRNAs associate with proteins to form snRNAs (small nuclear ribonucleoproteins) splicesosomes – como ribosomas pequenos, compuestos por RNA y proteinas Son 5 actuan en el nucleo U3 en el nucleolo para rRNA estan en forma snurps

RNA Splicing 2 pasos corte enel lado 5’ del intron formacion de enlace intramolecular entre G5’ y A2’ al lado 3’ requiere spliceosome completo y ATP formacion del lazo corte del lado 3’ y union de los dos exones 5’3’ Corte en el ado 5 ‘ intramolecular entre G5’ y A2’ al lado 3’ esta A es conservada

Key Points Noncoding intron sequences are excised from RNA transcripts in the nucleus prior to the transport to the cytoplasm. Introns in tRNA precursors are removed by the concerted action of a splicing endonuclease and ligase, whereas introns in some rRNA precursors are spliced out autocatalytically—with no catalytic protein involved.

Key Points The introns in nuclear pre-mRNAs are excised on complex ribonucleoprotein structures called spliceosomes. The intron excision process must be precise, with accuracy to the nucleotide level, to ensure that codons in exons distal to introns are read correctly during translation.