1.      What are the types and functions of RNA pol in eukaryotes?

RNA pol I- makes large ribosomal RNA

RNA pol II- transcribes RNA

RNA pol III-transcribes a variety of RNAs including transfer RNA and 5s ribosomal RNA

1.      What subunits form core RNA pol in bacteria?

2A, 1B,1B’, 1W

1.      What elements are found in prokaryotic promoters?

-10,-35,6+2 extended, UP element

1.      What is the function and subunit structure and function of the sigma factor?

a.       Sigma Factors function: to recognize different promoter sequences. IT has 3 sub units.

                                                              i.      Domain 2 binds to the -10 region, and helps separate duplex DNA (promoter melting).

                                                            ii.      Domain 3 recognizes the two bases of the extended -10 region

                                                          iii.      Domain 4 recognizes the -35 element – it is attached to a flexible part of the core enzyme which may let it accommodate different -35 to -10 spacing

1.      Describe regulation of flagella synthesis in salmonella

a.       While the proteins that form the flagellum base are being synthesized, the anti-sigma FlgM binds to σ^F preventing its binding to the core

b.       σ^F promotes transcription of genes needed for completion of flagellum assembly

c.        when base is assembled, FlgM is exported from the cell - σ^F transcribes late genes

1.      Describe assembly of pre initiation complex in eukaryotes

TFIID binds to TATA DCV

TFIIB is recruited binds BRE

TFIIA binds stabilizes TBP-DNA interactions

TFIIF core complex

TFIIE and TFIIH bind ATP powers unwinding of DNA

1.      What subunits and specific activity is required for transition from closed to open complex during eukaryotic protein coding genes transcription?

a.       Once RNA polymerase is in position, the complex of polymerase and promoter is called a closed complex

b.       RNA polymerase opens up about 14bp of duplex DNA (the transcription bubble)

c.        In Pol II, the transcription bubble is opened by helicase subunits of TFIIH and requires ATP

d.      The RNA polymerase bound to an open region of DNA is called the open complex

1.      What is abortive initiation?

Short RNAs of 2-9 nucleotides

1.      What causes abortive initiation?

Sigma loop 3.2 and beta finger of TFIIB

1.      What needs to happen for promoter clearance?

Displacement of the loop allows polymerase to break away from the promoter à promoter clearance

1.      What happens to RNA pol II as it converts from the initiating complex to the elongating complex?

·         The pre-initiation complex components are:

o   RNA polymerase II core enzyme

o   General transcription factors

·         In vivo transcription requires additional protein complexes that can alter the  chromatin structure (to remove histone barriers to transcription)

·         Another large complex, Mediator (which has more than 20 subunits), is needed to activate many Pol II transcribed genes

·         Once RNA polymerase is in position, the complex of polymerase and promoter is called a closed complex

·         RNA polymerase opens up about 14bp of duplex DNA (the transcription bubble)

·         In Pol II, the transcription bubble is opened by helicase subunits of TFIIH and requires ATP

·         The RNA polymerase bound to an open region of DNA is called the open complex

·         RNA polymerase frequently fails to make a full length RNA on the first attempt

·         This leads to release of short RNAs of 2-9 nucleotides, and is called abortive initiation

·         Once a transcription bubble is formed, RNA polymerase holds the template and non-template strands apart

·         The template strand binds to RNA polymerase at the active site

·         The non-template strand is held apart by regions called the “lid”, “zipper” and “rudder”

·         Ribonucleotides (NTPs) enter the active site and base pair with the template

·         Successive nucleotides are added at the 3′ end of the growing RNA molecule by nucleophilic attack, forming a phosphodiester bond and releasing pyrophosphate (this is very similar to DNA replication)

·         The 5′ end of the growing RNA leaves the polymerase via an exit channel

·         The loop blocks the elongating transcript

·         the loop must be moved in order for transcription to continue

·         Displacement of the loop allows polymerase to break away from the promoter – promoter clearance

·         RNA polymerase undergoes a conformational change that associates it very stably with DNA, and loosens its grip on initiation factors

·         Eukaryotic polymerase is phosphorylated as it converts to the elongating complex

1.      What types of RNA polymerases do Eukaryotes have?

RNA pol I- makes large ribosomal RNA

RNA pol II- transcribes RNA

RNA pol III-transcribes a variety of RNAs including transfer DNA and 5s ribosome RNA

1.      Difference between Transcription pausing and arrest

Pausing can restart and arrest is stopped for good

1.      3 factors that may cause pausing or arrest

•      the RNA transcript forms a hairpin

•       a weak DNA-RNA hybrid within the bubble caused by AU-rich sequence

•      base misincorporation happens)

Mechanism of transcript cleavage

a.       1) RNA polymerase backtracks

b.      2) 3′ end extrudes through the funnel

c.        3) Cleavage factors get into the funnel

d.       4) position a metal ion at the active site

e.        5) metal ion activates a water molecule for hydrolysis of the phosphodiester bond

1.      Describe the mechanism of transcription and capping coupling ( Not sure about this one)

a.       The capping enzyme binds to the 5’ end of transcription of modifie sit.

1.      What are two types of terminators?

a.       Intrinsic or simple

b.      Enzymatic terminators

c.       Rho-dependent terminators

1.      What are structural features of a simple terminator end transcription in the absence of other factors?

Inverted repeats from stem loops, string of A residues pairs with transcription poly-u

1.      What is the proposed mechanism of simple termination?

a.       Bacterial intrinsic terminators have:

b.       An inverted repeat that forms a stem-loop

c.       string of A residues - pairs with the transcribed poly-U - a weak structure)

d.       The weak base pairing in the transcription bubble is thought to arrest transcription

e.        the RNA hairpin helps to pull the RNA out of the active site

1.      What is the structure of the RHO protein?

Hexameric ATPase with a ring structure

1.      Describe a mechanism of RHO-dependent terminator

Binds with rut sites, RHO utilization, C-rich areas ring loads RNA ATP hydrolyzed brives pulling RNA

1.      Describe an allosteric model of RNA pol II

RNA pol II transcribes thru poly A and 3’ processing signals, RNA processes proteins associates with processing and signals and CTD cleavage processing proteins causing changes –( anitas answer is right hard to read)

1.      Describe a torpedo model of RNA pol II termination

Linked to degration of the cleavage RNA fragments RNA downstream of poly A sites to 3 endonucleases

1.      What are two types of protein structures used to regulate transcription?

Repressors and activators

1.      What are operators and what are their features?

Operators: bind and interact with regulatory proteins it is close to the promoter

1.      What are architectural proteins and their functions?

Architectural proteins are regulatory proteins that bind to turn loops to allow distal operators and enhancers to the interact

1.      What are enhancers and what are their features?

Enhancers are distal sequences in eukaryotes that interact with activator proteins

1.      What are insulators and how do they work in relation to gene expression?

Insulators: region that separate gene and enhancer

1.      What types of domains can transcription regulators have?

DNA bindind, aid oligmerization activate or repress transcription, interact with other regulators

1.      What are factors determining protein DNA binding?

Shape and charge

1.      Describe the structure of a helix- turn helix motif?

1^st helix is blue and the regonition helix dimmers, -alpha spirals a fixed distance 3.4 nm

1.      Describe the structure of a Zn motif

Has a domain of~30 amino acids with an alpha helix and 2 beta sheets

1.      Describe variations and structure of coiled coils motif

Sigma helices that wind around each other

1.      What is an operon?

The several genes needed to synthesis  trptophan are located together regulated together and transcribed together

What is lytic growth?

produces lots of copies of the virus in the bacterial cell and eventually leads to cell lysis

What is lysogeny?

the virus integrates into the host genome and becomes dormant (the integrated virus is a prophage)

1.      What is prophage?  Here are 2 definitions I found

                                                                                                  i.      is a phage (viral) genome inserted and integrated into the circular bacterial DNA chromosome. A prophage, also known as a temperate phage, is any virus in the lysogenic cycle; it is integrated into the host chromosome or exists a

                                                                                                ii.      The latent form of a bacteriophage in which viral genes are incorporated into bacterial chromosomes disrupting the bacterial cell. Also called probacteriophage

1.      How is lysogeny established by phage  lambda in E. Col

a.       regulated by levels of the DNA-binding proteins cI, cII and Cro

b.      The P_R promoter first produces Cro and cII

c.        If there is enough cII, it stimulates transcription at P_RE, which produces cI

d.       cI stimulates P_RM, which makes more cI.  High cI levels bind to P_R and P_L  and repress lytic genes, and also activates transcription of integrase 

e.       High cI levels promote and maintain lysogeny

f.        If not enough cII is produced, Cro binds to the P_RM promoter and represses cI production, ensuring expression of the lytic genes

g.       Levels of cII are crucial to establish lysogeny

1.      DNA damage

2.      Rec A protein produced

3.      Rec A destroys CI repressor

4.      Pr and PL promoters are activated

5.      CRO is produced

6.      CRO binds OR3 and blocks trabscription of CI from PRM

7.      PR and PL transcribe lytic genes / phages DNA disintegrates from the bacterial Chromosome.