Jos A. Card Serrano, PhDUniversidad Adventista de las AntillasBiol 223 GenticaAgosto 2010

Transfer of Genetic Information: The Central DogmaThe Process of Gene ExpressionTranscription in ProkaryotesTranscription and RNA Processing in EukaryotesInterrupted Genes in Eukaryotes: Exons and IntronsRemoval of Intron Sequences by RNA Splicing

The central dogma of biology is that information stored in DNA is transferred to RNA molecules during transcription and to proteins during translation.

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.

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.

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.

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.

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.

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

Method: Pulse-Chase LabelingAt 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.

Similar to DNA Synthesis exceptThe 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 53 direction.

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.

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.

Transcriptionthe first step in gene expressiontransfers the genetic information stored in DNAgenesinto messenger RNA molecules that carry the information to the ribosomesthe sites of protein synthesisin the cytoplasm.

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

Binding of RNA polymerase holoenzyme to a promoter region in DNALocalized unwinding of the two strands of DNA by RNA polymerase to provide a single-stranded templateFormation of phosphodiester bonds between the first few ribonucleotides in the anscent RNA chain

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.

Consensus sequences: -10 sequence and -35 sequenceRecognition sequence: -35 sequence

Rho-dependent terminatorsrequire a protein factor ()Rho-independent terminatorsdo not require

RNA synthesis occurs in three stages: (1) initiation, (2) elongation, and (3) termination.

RNA polymerasesthe enzymes that catalyze transcriptionare complex multimeric proteins.

The covalent extension of RNA chains occurs within locally unwound segments of DNA.

Chain elongation stops when RNA polymerase encounters a transcription-termination signal.

Transcription, translocation, and degradation of mRNA molecules often occur simultaneously in prokaryotes.

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.

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.

TFIID se une a TATA boxTFIID contiene TBP

TFIIA se une al complejo

TfIIB

Dnase Fingerprinting

TFIIF helicasa - iniciacion- se asocia primero a la pol y luego ambos al complejo

Polimerasa II

TFIIE

TFIIH y TFIIJ-ubicacion desconocidaH helicasa para extension

- Observar los zurcos donde se enlaza el DNA y donde se libera el RNA nasciente

1ra modificacion del preMRNA

adicion de guanosinsa metilada

enlace 5-5

se anade cotranscripcional

2 funciones iniciacion de traduccion estabilidad/proteccion

Terminacin - -por corte endonucleolitico del pre mRNAvarios puntos, 1000-2000 down de donde sera el 3AAUAA y GU poliA pol aade 200 A2 funciones:estabilidadtransporte

Usually the genetic information is not altered in the mRNA intermediary.

Sometimes RNA editing changes the information content of genes byChanging the structures of individual basesInserting or deleting uridine monophosphate residues.

Edicion de modificar CU4563 aa vs 2153 aaUAA Codon de terminacion prematuroDeaminacion de CInsercion de bases usando los RNA guiasmitocondrias y Tripanosomas

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, andThe excision of noncoding intron sequences.

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

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.

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.

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.

The noncoding introns are excised from gene transcripts by several different mechanisms.

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-GTAG-exonTACTAAC box about 30 nucleotides upstream from the 3 splice site.

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.

RNA/protein structureExcises introns from nuclear pre-mRNA

Five snRNAs: U1, U2, U4, U5, and U6Some snRNAs associate with proteins to form snRNAs (small nuclear ribonucleoproteins)

2 pasoscorte enel lado 5 del intronformacion de enlace intramolecular entre G5 y A2 al lado 3requiere spliceosome completo y ATPformacion del lazocorte del lado 3 y union de los dos exones 53

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 autocatalyticallywith no catalytic protein involved.

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.

*El mRNA es como un diamante sin pulir, explique:Computadoras usan codigo binarios de 0 y 1Clave morse usa codigo de puntos y rayas, dos simbolosAlfabeto 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**********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..

*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

**DiferenciasEnfasis en Sense y nonsense 0 non template y template:

*Direccion de la sintesis de RNA ocurre 53*Promotores secuencias especificas donde se pega la po.imerasajunto 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****Transcripcion es basicamente igual en procariotes y eucariotes pero tambien hay diferencias

Unidad transcripcional un segmento de DNA que es transcrito pueden ser genes individualesen 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*RNA pol enzimas multimericasla de E coli tiene 5 polipeptidos 2 son iguales por lo tanto tienen 4 polipeptidosHoloenzima 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

*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

**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

*2do paso es alargamiento Catalizada por el core de la RNA pol (sin sigma)La RNA Pol tiene actividad de enrollar y desenrollarabre un area como de 18 ntd y anade 40 nct por segsolo pareo de 3 bases en el hibridoy la estabilidda se mantiene porq ambas cadenas estan unidas a la RNA POl*Para terminacion hay una senal que la RNA pol encuentra y reconoceal llegar ahi el complejo se disociasuelta la cadena naciente

rho - u**En procario los dos procesos son acopladostranscripcion y traduccion y degradacion al mismo tiempo sobre una misma hebra de DNAlos tres procesos ocurren 53

En procariotes no hay compartamentalizacion

***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 eucariotesExcepction 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.

*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*3 polimerasas para 3 sets de genes

Requieren factorres de transcripcion distinto al a de procariotas

Distintas sensibilidades a alfa amanitinaRNA 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*Una RNA pol II tipica una RNA pol dependiente de DNAtranscribe genes estructiralesdesenredar la doble helice con la ayuda de TF q se pegan a secuencias especificaspromotor visto aqui distintas secuenciasTATAAAA y GGCCAATCT elementos de secuencias conservados -30 y -80GC boxReconocer iniciar efeiciencia

*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 vanletra va con orden de descubrimiento

Donde se pega cada factor enel DNA se determina con exptos de proteccion de degradacion en I termedios Dnase fingerprintingoligos con la secuecia putatitva marcados radioactivamente en un extremoincubar la mitad con la proteina de interes o el TF y la otra noTratarlo scon DNAsa pancreatica I. brevemente para solo un corte (hace cortes al azar en el dna)Electroforesisla muestra sin proteinas tendra pedazos de todos los tamanos posiblesLa muestra con las proteinas tendra pedazos similares y algun pedazo faltara o sera mas gdela proteina no dejo q lo cortaran PLT esa secuencia fue protegina

*Las RNA pol I y II realizan procesos similares con pequenas diferenciasmas 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*Una vez la polimerasa se suelta del complejo de iniciacion, sigue con el alargamientoPor un lado se une al DNA templado y por otro lado sale la molecula nueva

*Temprano en el proceso cuando van solo 30 nct

*FACT para evadir nucleosomasconvierte octamero en hexameros remuven H2A/H2Bhistonas acetiladas tambien facilintan

**Ejemplo de Editing de mRNA o del intermediario

***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 proteinasIntronesExones

La mayoria de los genes tienen intronesEJ VTG de Xenopus tiene 33 intrones

Colageno de ave tiene 50 intrones resultado: ungen de 37,000 da un mRNA de 4600 nct*Heteroduplex de mRNA y DNA del genoma de Adenovirus

***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****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

*Corte en el ado 5 intramolecular entre G5 y A2 al lado 3esta A es conservada

**