Capítulo 13 Microbial Genetics Central Dogma Regulation of Gene Expression Genetic Exchange R E P L I C A C I Ó N Y E X P R E S I Ó N D E L G E N O M A B A C T E R I A N O Dogma Central de la Genética 5 3 3 5 DNA (genotype) Transcription 5 Translation by ribosomes mrna 3 NH 2 Methionine Arginine Tyrosine Leucine Polypeptide Phenotype 1
Flujo de la Información Genética DNA M A T E R I A L G E N É T I C O Experimentos De: Transformación Griffith Avery, MacCartey, McLeod Hershey & Chase 2
Estructura de los Á C I D O S N U C L E I C O S Y P R O T E Í N A S 3
Genoma Bacteriano Precursors and Building Blocks of Nucleic Acids Nucleotides polymerize to a nucleic acid by phosphodiester bonds. An organic ester: R-C-O-C-R A phosphodiester: =O =O R-C-O-P-O-C-R =O 4
Base Pair Complementation Pyrimidines: C & T (U in RNA) Purines: G &A G to C by 3 H-bonds A to T (U) by 2 H-bonds High G+C content of DNA means more H-bonds; requires more energy to separate (denature) duplex. 5 -P 3 -OH DNA Structure: Complementation of Two Antiparallel Strands Replicación del DNA E N L A S B A C T E R I A S Forms into a Double Helix Conformation 3 -OH 5 -P 5
DNA Replication Overview * Eucariotas It is semi-conservative; half from the parent half newly synthesized. It s initiated at a replication fork; DNA must be unwound and unbound into two single strands. Parent strands become the templates for the new (complementary) strands. Enzyme Functions in Replication DNA Polymerase III This is a simple cartoon; exaggerated to illustrate function. DNA Polymerase Reaction It must add to an existing nucleic acid (RNA or DNA). Adds dntp to the 3 -OH end of a new strand; it grows in the 5 3 direction. Ligase (connects O- fragments) Primosome (makes RNA primer) DNA Polymerase I (also has RNase, 5 3 exonuclease, activity to remove RNA primer) DNA-Poly III has Proofreading capability; can catch and fix mistakes most of the time by 3 5 exonuclease activity. 6
Replisome Two DNA Polymerase III stay together in a complex called the replisome. This requires the lagging strand to loop back onto its strands DNA Polymerase III. When the lagging strand DNA Polymerase reaches the previous Okazaki Fragment it drops off and then grabs the next RNA primer. Replicación del Cromosoma Bacteriano (OriC) Termination (ter) Prokaryote Chromosome Replication (OriC) Iniciación de la Replicación Chromosome is of circular DNA, and supercoiled (extra twists in helix knotsup or condenses DNA when not replicating). Plasmids = extrachromosomal element; circular; 100-1000 times smaller than chromosome. Also supercoils when not replicating. Plasmid DNA replication like that of chromosome or by rolling circle replication (more later in discussing bacterial conjugation, sex ). Termination (ter) ~30 Replisoma 7
Replication Initiation Initiator proteins (IP; about 30) bind at repetitive sequences within the OriC site. DNA winds around IP-complex; induces separation of strand at adjacent AT rich site. Single strand binding proteins & helicases attach. Formation of two replication forks that open in opposite directions. ~30 Transcripción Transcripción D N A R N A Definición síntesis de RNA bajo la dirección del DNA Requisitos Cadena molde de DNA o cadena con sentido (template strand) Enzimas RNA polimerasa - DNA dependiente (DNA directed) α ensamblaje del núcleo, reconocimiento de los promotores, interacción con los factores reguladores. β se une a los sustratos ribonucleótidos β se encuentra en el sitio de unión con el DNA ώ σ ayuda al núcleo de la enzima a reconocer el comienzo de los genes y a la RNA pol. a unirse con la región promotora. 8
RNA Polimerasas RNA pol. I - cataliza la síntesis de rrna (5.85S, 18S,28S) RNA pol. II cataliza la síntesis de mrna RNA pol III cataliza la síntesis de trna y rrna 5S Transcription Genes + p/operator = operon. Sigma-factor 3 5 Operator region Gene(s) 5 antisense 3 sense Antisense (template) strand Sense (complementary) strand Trailer sequence 9
Requisitos cont. Eventos Sustratos ATP UTP GTP CTP Iones minerales Zn Mg Factores Iniciación (IF) Alargamiento (EF) Terminación (TF) Secuencia líder Pirofosfato Iniciación La enzima RNA polimerasa reconoce las regiones de consenso TTGACA, TATAAT (caja de Pribnow). La RNA polimerasa se une a la región promotora. El RNA se transcribe downstream comenzando en +1. La RNA polimerasa abre y desenrolla la DS DNA. Se forma la burbuja de transcripción El primer nucleótido incorporado es ATP o GTP. Alargamiento La RNA polimerasa sintetiza una cadena de nueve (9) nucleótidos de longitud. Región Promotora Sitio de reconocimiento y acoplamiento de la RNA pol Reconocimiento requieres un factor sigma Diferentes promotores tienen su propio factor sigma Puede haber un sitio de acoplamiento para el regulador ( = operador) adyacente. Pribnow Box 5 3 mrna Elongation (recall that Us replace Ts) 5 3 10
Terminación Eventos Secuencias terminadoras en el DNA contienen palíndromes. Señales de terminación rho independientes de rho Transcripción de los palíndromes Formación de una horquilla RNA polimerasa reduce la velocidad de transcripción, se para completamente Procesamiento del RNA Deaminación, metilación, reducción Transcription Termination Rho dependent: a protein called ρ unwinds the DNA template mrna complex; thus weakening the association until mrna and RNA polymerase falls off DNA. Rho-Independent: here two G+C regions rich slow RNA polymerase; a hairpin forms in the mrna weakening the mrna-dna template association; which is further weakened by poly-a stretch. rrna and trna Products rrn-gene has multiple rrna & trna products. Spacer RNA is spliced out by special nucleases. rrn-operons are often located close to Ori. Why might this have been selected? (think fast growth)? mrna product Within the leader is the Shine-Dalgarno sequence (5 AGGA3 ). It complements a site on 16SrRNA of ribosome; used to bind a ribosome to mrna for translation. Trailer at 3 end of mrna was required for proper ending of transcription. Translation of mrna information into protein starts at the initiation codon (5 AUG3 ); proceeds codon by codon until the end of gene, as identified by a stop, or non-sense, codon. Coding region of mrna can be polygenic (multiple genes = multiple starts / stops). Coding Region Direction of Translation 11
Terminación de la Transcripción Dependiente de Rho: una proteína llamada ρ desenrrolla el templado DNA mrna; así se debilita la asociación hasta que mrna y RNA pol se sueltan o se desprenden (falls off) del DNA. Rho-independente: aquí dos regiones ricas en G+C hacen que la RNA polimerasa disminuya la velocidad; se forma una horquilla en el mrna. debilitando la asociación mrna DNA templado; la cual se debilita aún más cuando poly-a se estira. Procesamiento del mrna en los Eucariotas 12