Del ADN a la proteína: expresión génica

Dogma central de la biología molecular Transcripción Reversa Replicación Transcripción Traducción ADN ARN Proteína No hay flujo de información de la proteína al ARN o al ADN Propuesto por Francis Crick

Dogma central de la biología molecular No hay flujo de información de la proteína al ARN o al ADN Propuesto por Francis Crick

Donde estan las proteínas? De que nos sirve saber que genes codifican para cada proteína? Podríamos controlar el metabolismo, e.g., aumentando la síntesis de una enzima e impidiendo la síntesis de otra.

Diferencias entre al ADN y el ARN ADN doble helice, ARN usualmente una solo habra, que se puede plegar sobre si misma Se pueden formar hibridos de ADN-ARN

Diferencias entre al ADN y el ARN ADN doble helice, ARN usualmente una solo habra, que se puede plegar sobre si misma. Timina es reemplazada por uracilo en el ARN

El ARN se puede plegar sobre si mismo – estructura secundaria La estructura secundaria de muchos ARNs es importante para que desempeñen sus función biológica. Origen del video The double life of RNA DVD. HHMI.

Principales tipos de ARN ARNt La estructura secundaria de muchos ARNs es importante para que desempeñen sus función biológica. Ribosomal mRNA

Figure 14.2 From Gene to Protein

Transcripción de ARN: RNA polimerasa No necesita primers No tiene función deprueba de lectura

Reconocimiento de señales de inicio del gen en el promotor Iniciación de la transcripción Reconocimiento de señales de inicio del gen en el promotor Se necesita un promotor, regiones especiales el ADN que señalan que el ADN debe ser transcrito La ARN polimerasa se une al promotor El sitio donde inicia la transcripción, que hace parte del promotor, se llama el sitio de inicio de la transcripción El promotor indica donde empezar y que hebra transcribir

Elongación y terminación de la transcripción Elongation: RNA polymerase unwinds DNA about ten base pairs at a time; reads template in 3′ to 5′ direction. The RNA transcript is antiparallel to the DNA template strand, and adds nucleotides to its 3′ end. RNA polymerases do not proofread and correct mistakes. Termination: Is specified by a specific DNA base sequence. Mechanisms of termination are complex and varied. For some genes the transcript falls away from the DNA template and RNA polymerase—for others a helper protein pulls it away. No hay corrección de errores, horquilla de 10nt

Transcripción del ARN Each eukaryote gene has one promoter to which RNA polymerase binds, with the help of other molecules. At the other end of the gene there is a terminator sequence to signal end of transcription.

El código genético es degenerado pero no ambiguo The genetic code: Specifies which amino acids will be used to build a protein Codon: A sequence of three bases—each codon specifies a particular amino acid. Start codon: AUG—initiation signal for translation. Stop codons: UAA, UAG, UGA—stop translation and polypeptide is released. For most amino acids, there is more than one codon; the genetic code is redundant. The genetic code is not ambiguous—each codon specifies only one amino acid.

Dogma central de la biología molecular Transcripción Reversa Replicación Transcripción Traducción ADN ARN Proteína No hay flujo de información de la proteína al ARN o al ADN Propuesto por Francis Crick

Reconocimiento de señales de inicio del gen en el promotor Iniciación de la transcripción Reconocimiento de señales de inicio del gen en el promotor Se necesita un promotor, regiones especiales el ADN que señalan que el ADN debe ser transcrito La ARN polimerasa se une al promotor El sitio donde inicia la transcripción, que hace parte del promotor, se llama el sitio de inicio de la transcripción El promotor indica donde empezar y que hebra transcribir

Elongación y terminación de la transcripción Elongation: RNA polymerase unwinds DNA about ten base pairs at a time; reads template in 3′ to 5′ direction. The RNA transcript is antiparallel to the DNA template strand, and adds nucleotides to its 3′ end. RNA polymerases do not proofread and correct mistakes. Termination: Is specified by a specific DNA base sequence. Mechanisms of termination are complex and varied. For some genes the transcript falls away from the DNA template and RNA polymerase—for others a helper protein pulls it away. No hay corrección de errores, horquilla de 10nt

La presencia de intrones es común en genes eucarióticos Estructura de los genes eucarioticos, hay regiones no codificantes Eukaryotic genes may have noncoding sequences—introns. The coding sequences are exons. Introns and exons appear in the primary mRNA transcript—pre-mRNA; introns are removed from the final mRNA. La presencia de intrones es común en genes eucarióticos La inmensa mayoría de los procariotes no tienen intrones

Estructura de los genes en eucariotes Introns interrupt, but do not scramble, the DNA sequence that encodes a polypeptide. Sometimes, the separated exons code for different domains (functional regions) of the protein. En algunos casos los exones codifican para dominios independientes de las proteínas, funcionan como

ARN splicing: corte y empalme del ARNm RNA splicing removes introns and splices exons together. Newly transcribed pre-mRNA is bound at ends by snRNPs—small nuclear ribonucleoprotein particles. Consensus sequences are short sequences between exons and introns. snRNPs binds here, and also near the 3′ end of the intron. With energy from ATP, proteins are added to form an RNA-protein complex, the spliceosome. The complex cuts pre-mRNA, releases introns, and splices exons together to produce mature mRNA.

Modificaciones en el ARNm maduro In the nucleus, pre-mRNA is modified at both ends: G cap is added at the 5′ end (modified guanosine triphosphate)—facilitates mRNA binding to ribosome. G cap protects mRNA from being digested by ribonucleases. Poly A tail added at 3′ end. AAUAAA sequence after last codon is a signal for an enzyme to cut the pre-mRNA; then another enzyme adds 100 to 300 adenines—the “tail.” May assist in export from nucleus; important for stability of mRNA.

Modificaciones en el ARNm maduro: Capping Adicion de una Guanosina metilada al extremo 5’ del ARNm. El enlace es entre dos carbonos 5 y es un puente tri-fosfato. http://en.wikipedia.org/wiki/File:5'_cap_structure.png

Modificaciones en el ARNm maduro El ARN mensajero maduro tiene que ser exportado del núcleo al citoplasma a través del poro nuclear en un proceso mediado por proteínas. Una vez en el citoplasma el ARNm puede ser traducido In the nucleus, pre-mRNA is modified at both ends: G cap is added at the 5′ end (modified guanosine triphosphate)—facilitates mRNA binding to ribosome. G cap protects mRNA from being digested by ribonucleases. Poly A tail added at 3′ end. AAUAAA sequence after last codon is a signal for an enzyme to cut the pre-mRNA; then another enzyme adds 100 to 300 adenines—the “tail.” May assist in export from nucleus; important for stability of mRNA.

ARN de transferencia tRNA, the adapter molecule, links information in mRNA codons with specific amino acids. For each amino acid, there is a specific type or “species” of tRNA. Two key events must happen to ensure that the protein made is the one specified by the mRNA: tRNAs must read mRNA codons correctly tRNAs must deliver amino acids corresponding to each codon Three functions of tRNA: It binds to an amino acid, and is then “charged” It associates with mRNA molecules It interacts with ribosomes

¿Cómo funcional el codon y el antocodon? Example: DNA codon for arginine: 3′-GCC-5′ Complementary mRNA: 5′-CGG-3′ Anticodon on the tRNA: 3′-GCC-5′. This tRNA is charged with arginine.

El código genético The genetic code: Specifies which amino acids will be used to build a protein Codon: A sequence of three bases—each codon specifies a particular amino acid. Start codon: AUG—initiation signal for translation. Stop codons: UAA, UAG, UGA—stop translation and polypeptide is released. For most amino acids, there is more than one codon; the genetic code is redundant. The genetic code is not ambiguous—each codon specifies only one amino acid. Wobble: Specificity for the base at the 3′ end of the codon is not always observed. Example: Codons for alanine—GCA, GCC, and GCU—are recognized by the same tRNA. Wobble allows cells to produce fewer tRNA species, but does not allow the genetic code to be ambiguous.

Cargando las moléculas de ARNt Activating enzymes—aminoacyl-tRNA synthetases—charge tRNA with the correct amino acids. Each enzyme is highly specific for one amino acid and its corresponding tRNA; the process of tRNA charging is called the second genetic code. The enzymes have three-part active sites: They bind a specific amino acid, a specific tRNA, and ATP.

Ribosomas Activating enzymes—aminoacyl-tRNA synthetases—charge tRNA with the correct amino acids. Each enzyme is highly specific for one amino acid and its corresponding tRNA; the process of tRNA charging is called the second genetic code. The enzymes have three-part active sites: They bind a specific amino acid, a specific tRNA, and ATP.

Figure 14.14 Ribosome Structure Aminoacil Salida (Exit) Peptidil Ribosome: the workbench—holds mRNA and charged tRNAs in the correct positions to allow assembly of polypeptide chain. Ribosomes are not specific, they can make any type of protein. Ribosomes have two subunits, large and small. In eukaryotes, the large subunit has three molecules of ribosomal RNA (rRNA) and 49 different proteins in a precise pattern. The small subunit has one rRNA and 33 proteins. Ribosomal subunits are held together by ionic and hydrophobic forces (not covalent bonds). When not active in translation, the subunits exist separately. Large subunit has three tRNA binding sites: A (amino acid) site binds with anticodon of charged tRNA P (polypeptide) site is where tRNA adds its amino acid to the growing chain E (exit) site is where tRNA sits before being released from the ribosome. Ribosome has a fidelity function: When proper binding occurs, hydrogen bonds form between the base pairs. Small subunit rRNA validates the match—if hydrogen bonds have not formed between all three base pairs, the tRNA must be an incorrect match for that codon and the tRNA is rejected.

Iniciación de la traduccón An initiation complex forms—a charged tRNA and small ribosomal subunit, both bound to mRNA. In prokaryotes rRNA binds to mRNA recognition site “upstream” from start codon. In eukaryotes the small subunit binds to the 5′ cap on the mRNA and moves until it reaches the start codon. mRNA start codon is AUG: First amino acid is always methionine, which may be removed after translation. The large subunit joins the complex; the charged tRNA is now in the P site of the large subunit. Initiation factors are responsible for assembly of the initiation complex When the first tRNA has released its methionine, it moves to the E site and dissociates from the ribosome—can then become charged again. Elongation occurs as the steps are repeated, assisted by proteins called elongation factors.

Sitios de unión del ribosoma http://www.ambion.com/techlib/append/rbs_requirements.html

Elongación de la traducción Elongation: The second charged tRNA enters the A site. Large subunit catalyzes two reactions: It breaks bond between tRNA in P site and its amino acid Peptide bond forms between that amino acid and the amino acid on tRNA in the A site

Elongación de la traducción

Elongación de la traducción

Figure 14.17 The Termination of Translation (Part 1) Termination: Translation ends when a stop codon enters the A site. Stop codon binds a protein release factor—allows hydrolysis of bond between polypeptide chain and tRNA on the P site. Polypeptide chain separates from the ribosome—C terminus is the last amino acid added.

Figure 14.17 The Termination of Translation (Part 2)

Figure 14.17 The Termination of Translation (Part 3)

Traducción Prokaryotes and eukaryotes differ in gene structure—in the organization of nucleotide sequences. In eukaryotes a nucleus separates transcription and translation.

Diferencias entre procariotes y eucariotes a nivel de la transcripción Prokaryotes and eukaryotes differ in gene structure—in the organization of nucleotide sequences. In eukaryotes a nucleus separates transcription and translation.

Figure 14.18 A Polysome (A)

Figure 14.18 A Polysome (B)

Figure 14.19 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell

Figure 14.21 A Signal Sequence Moves a Polypeptide into the ER (2)