Understanding RNA Polymerase Initiation and Operon Structure

2. 3´ 5´ 3´ 5´ +1 Promotores Secuencias de ADN que posicionan a la RNA polimerasa en el sitio de iniciación

3. RNA polymerase binds to specific promoter sequences to initiate transcription Each subunit has a specific function

4. s70 Most genes TTGACAT TATAAT s32 Genes induced by heat shock TCTCNCCCTTGAA CCCCATNTA s28 Genes for motility and chemotaxis CTAAA CCGATAT s38 Genes for stationary phase and stress response ? ? s54 Genes for nitrogen metabolism and other functions CTGGNA TTGCA The s subunit contributes to specific initiation • It decreases the affinity of RNA polymerase for general regions of DNA by a factor of 104 • Enables RNA polymerase to recognize promoter sites this helix has been implicated in recognizing the 5-TATAAT sequence of the -10 region • the promoter site is encountered by a random walk in one dimension rather than in three dimensions. • The s subunit is released when the nascent RNA chain reaches nine or ten nucleotides in length. After its release, it can assist initiation by another core enzyme. • Thus, the s subunit acts catalytically.

5. An Operon Consists of Regulatory Elements and Protein-Encoding Genes (A)The general structure of an operon as conceived by Jacob and Monod. (B) The structure of the lactose operon. In addition to the promoter (p) in the operon, a second promoter is present in front of the regulator gene (i) to drive the synthesis of the regulator.

6. • lacZ codes for the enzyme β-galactosidase, tetramer of-500 kD. The enzyme breaks a β-galactoside into its component sugars. • lacYcodes for the β-galactoside permease, a 30 kD membrane-bound protein This transports β-galactosides into the cell. • lac Acodes for β-galactoside transacetylase, an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides.

8. THE LAC OPERATOR SEQUENCE IS A NEARLY PERFECT INVERTED REPEAT CENTERED AROUND THE GC BASE PAIR AT POSITION + 11 The 17-bp sequence of the top strand beginning at −7 is identical to the 17-bp sequence of the bottom strand beginning at +28, reading in the 5′ → 3′ direction in both cases, except for the nucleotides indicated by italic letters. Each half of the inverted-repeat sequence is called an operator half-site (yellow highlight).

12. Cooperative binding of cAMP-CAP and RNA polymerase to the lac contol region activates transcription

14. Se han obtenido varios mutantes en la expresión de la ß-galactosidasa. En la tabla se indican las unidades relativas de esta enzima producidas por los distintos mutantes en varias condiciones de inducción: • W es la estirpe no mutante. • Si a M3 si le añade IPTG (inductor que no requiere permeasa para entrar) en vez de lactosa, los niveles de expresión son idénticos a los de la estirpe W. • En M4, la utilización de IPTG en lugar de lactosa no modifica los resultados • En M5 los niveles de mRNA del operón son similares a los de W. En cambio, en M6 no se detecta mRNA del operón en las diversas condiciones de inducción. • Determine para cada cepa mutante en dónde se encuentra la lesión genética. Explique el razonamiento que le permitió arribar a esas conclusiones.

15. Experimental evidence for trans-acting genes/proteins Experimental evidence for cis-acting DNA sequences

16. Disponemos de cinco cepas de E. coli conteniendo los siguientes merodiploides: a) I-P+O+Z+Y+A+/I+P+O+Z-Y+A+ b) I-P+OcZ+Y+A-/I+P+O+Z-Y+A+ c) IsP+O+Z+Y+A+/I-P+O+Z-Y+A+ d) I-P-O+Z+Y+A+/I-P+OcZ+Y-A+ e) I-P+O+Z-Y+A+/I-P-OcZ+Y-A+ Donde I indica el gen del represor lac; P y O el promotor y operador, respectivamente. lac Z, Y, A, genes estructurales del operón lac codificando la b-galactosidasa, permeasa y transacetilasa, respectivamente. Cada una de estas cepas se cultivó en un medio sin lactosa (ni glucosa, o con glicerol como fuente de C) y luego se añadió lactosa como única fuente de carbono. Represente para cada cepa una gráfica de la concentración de cada enzima (b-galactosidasa Z, permeasa Y y transacetilasa A) (eje y) en función del tiempo (eje x). Las concentraciones se midieron durante 30 min empezando 10 min antes de añadir lactosa.

17. Dual Positive And Negative Control: the Arabinose Operon

18. araO1 is an operator site. AraC binds to this site and represses its own transcription from the PC promoter. In the presence of arabinose, however, AraC bound at this site helps to activate expression of the PBAD promoter. araO2 is also an operator site. AraC bound at this site can simultaneously bind to the araI site to repress transcription from the PBAD promoter araI is also the inducer site. AraC bound at this site can simultaneously bind to the araO2 site to repress transcription from the PBAD promoter. In the presence of arabinose, however, AraC bound at this site helps to activate expression of the PBAD promoter. CRP binds to the CRP binding site. It does not directly assist RNA polymerase to bind to the promoter in this case. Instead, in the presence of arabinose, it promotes the rearrangement of AraC when arabinose is present from a state in which it represses transcription of the PBAD promoter to one in which it activates transcription of the PBAD promoter.

19. When arabinose is absent AraC binds simultaneously to araI and araO2. As a result the intervening DNA is looped. These two events block access to the PBAD promoter which is, in any case, a very weak promoter When arabinose is present, it binds to AraC and allosterically induces it to bind to araI instead araO2. If glucose is also absent, then the presence of CRP bound to its site between araO1 and araI helps to break the DNA loop and also helps AraC to bind to araI:

20. Dual Positive And Negative Control: the Arabinose Operon

21. Additional examples of control attenuation

26. Activation of 54-containing RNA polymerase at glnA promotor by NtrC Necesita ATP para formar el complejo abierto!!

27. Organisms use RNA in a wide range of regulatory mechanisms to control gene expression. The classical examples of such regulation are transcription and translation attenuation in bacteria In this review, we describe a new class of regulatory RNA that needs the same principles of alternative structure formation to control transcription elongation and translation initiation depending on the metabolic status of the cell. The uniqueness of these RNA systems is that they do not require any intermediary sensory molecules (i.e. protein factors or tRNA) to govern the attenuation process; they behave as sensors of small molecules themselves

28. artificial smallmolecule-bindingRNAscalledaptamers

30. Many bacterial responses are controlled by two-component regulatory systems The PhoR/PhoB two-component regulatory system in E. coli

31. Agrobacteriumtumefaciens Crown gall tumor