Chapter 15 DNA-Dependent Synthesis of RNA

What is the RNA polymerase core?

A large complex enzyme in E. coli. It has five subunits: Beta, beta prime, alpha (two copies), and omega (pg. 519)

What is the mediator complex?

A large, multiprotein complex in eukaryotic cells that serves as the mediator between the Pol II transcription complex and any upstream transcription activators or enhancers regulating Pol II-catalyzed transcription. Think of it like a BRIDGE (pg. 538)

What is DNA footprinting?

A method of investigating the sequence specificity of DNA-binding proteins in vitro. This technique can be used to study protein-DNA interactions both outside and within cells. In this experiment, DNA footprinting shows which sigma factor binds to a promoter in response to osmotic shock. Sigma-38 directs RNA polymerase to genes that respond to cellular stresses so this is expressed more when we increase osmotic shock. Sigma-70 is for "housekeeping" genes so it's used less with increasing osmotic shock (pg. 527)

What are transcription factors?

In eukaryotes, a protein that affects the regulation and transcription initiation of a gene by binding to a regulatory sequence near or within the gene and interacting with RNA polymerase and/or other transcription factors (pg. 532)

What is the closed complex in context of transcription? What about the open complex?

The closed complex is when the DNA bound to RNA polymerase is still intact (not separated). The open complex is when the DNA is separated (opened). (pg. 521)

What is transcription?

The enzymatic production of an exact complementary strand of RNA on a DNA template (pg. 516)

What is mRNA? rRNA?, tRNA?

mRNA is messenger RNA and it encodes the amino acid sequence of one or more polypeptides specified by a gene or a set of genes. rRNA are constituents of ribosomes tRNA read the information encoded in the mRNA and provide the appropriate amino acid to a growing polypeptide chain during protein synthesis.

What three types of RNA is formed from transcription?

mRNA, rRNA, and tRNA

What are the three phases of transcription?

1) Initiation: RNA polymerase binds to the template strand 2) Elongation: the process of adding nucleotides to the growing RNA strand 3) Termination: the release of the product RNA whent he polymerase reaches the end of a gene or other transcription unit (pg. 519)

What is the transcription cycle for RNA pol II promoters?

1) Pol II recruitment by transcription factors 2) Formation of transcription bubble 3) Phosphorylation of CTD during initiation 4) Elongation 5) Termination pg. 535-536

What is osmotic shock?

A sudden change in the solute concentration around a cell, causing a rapid change in the movement of water across its cell membrane. Google

Differentiate between RNA pol I, II, and III. Which one is responsible for transcribing protein-coding genes

All of these are found in eukaryotes. Pol I if for rRNA. Pol II is for proteins. Pol III is for smaller functional RNAs like tRNAs. RNA pol II is responsible for transcribing protein-coding genes (pg. 519)

DNA footprinting and Sp1 protein binding

As we increase the amount of Sp1, the sites that it binds to on the DNA become more pronounced. The "footprint" becomes more pronounced. (pg. 541)

The chemical mechanism for RNA synthesis

Basically RNA polymerase uses a DNA template strand to transcribe complementary RNA. Remember that RNA uses uracil instead of thymine. Notice how the RNA is made in the 5' to 3' direction.

What subunits make up the RNA polymerase core in bacteria?

Beta, beta prime, alpha (two copies), and omega

True or false: all promoters have the same sequence.

False, there is variation in promoter sequences. In general, the more a promoter exhibits the consensus sequence, the stronger it is

What's different about the RNA polymerases in bacteria and Eukaryotes?

In bacteria there is a single RNA polymerase that transcribes the genes to make mRNA, tRNA, and rRNA. In Eukaryotes, there are three different RNA polymerases that each transcribe for rRNA, tRNA, and rRNA respectively

Describe the typical eukaryotic RNA polymerase I promoter.

It has an upstream control element (UCE) and a core sequence. UBF binds both of UCE's. SL1 binds the UBFs and Pol I allowing for transcription (pg. 534)

What is the sigma factor of the RNA polymerase core?

It's a sixth subunit in Bacterial of the RNA polymerase core that binds transiently to the RNA polymerase core and directs the enzyme to specific binding sites on DNA. IT PROVIDES PROMOTER SPECIFICITY (pg. 519)

What is the consensus sequence?

It's like the 'average' of all the possible promoter sequences. (pg. 524)

Sigma factors and strand opening

Most sigma factors allow RNA polymerase to open the strand without ATP. (pg. 528)

Discuss the proofreading of RNA polymerase. Two ways?

RNA polymerase can proofread in two ways: kinetic proofreading and nucleolytic proofreading. In kinetic proofreading, we remove the base before proceeding in transcription. In nucleolytic proofreading, we remove a short stretch of bases

Table of E. Coli sigma factors

Sigma 70 is for the "housekeeping" genes epressed in all growing cells (pg. 525)

What are promoters?

Specific DNA sequences that initiate RNA transcription (pg. 519)

Which of the following -10 sequences found in promoters of bacterial genes would you predict to be the 'strongest?' TATTAT TATCAT TAGCAT TAGAAG TCGAAT

TATTAT

What is the core promoter?

The DNA sequence elements in eukaryotic cells common to promoters used by Pol II. The TATA box and initator sequence (Inr) are required elements of a core promoter; a TFIIB recognition element (BRE) and downstream promoter element (DPE) may also be involved in transcription initiation from some core promoters (pg. 534)

Describe the transcription "bubble"

The part of the DNA that is unwound allowing for transcription. Supercoiling occurs in front of and behind the transcription bubble (pg. 520)

In what direction is RNA synthesized?

The same direction that DNA is synthesized: 5' to 3'. (The template strand is copied in the 3' to 5' direction)

Differentiate between template strand, nontemplate strand, and the coding strand.

The template strand is the one bound to the RNA polymerase it has a complementary sequence to the RNA strand. The nontemplate strand and coding strand are the same thing. It has an identical sequence (except for Ts instead of Us) as the RNA strand (pg. 520)

Channels in RNA polymerase

There's an enter and exit channel for the DNA, an rNTP entry channel, and an RNA exit channel (pg. 528)

Comparison of bacterial RNA polymerase and eukaryotic RNA polymerase II.

They look similar, but the Eukaryotic pol II is more complex because eukaryotes are more complicated than bacteria (pg. 536)

How is transcription halted in bacteria?

Transcription stops when the RNA polymerase transcribes through TERMINATION SEQUENCES. There are two termination sequence classes: rho-independent (requires a stem-loop) or rho factor dependent (rho is a helicase) (pg. 531)

Bacterial promoters

We have an upstream promoter element (AT rich) and a spacer between the -35 region and -10 region (pg. 524)

Give an overview of transcription.

We start with duplex DNA with a promoter. RNA polyerase binds to the DNA promoter. We then form the transcription bubble. Initiation, elongation (AKA promoter clearance), and finally termination (and recycling) (pg. 521)

What is mutation analysis? Explain how mutational analysis allows identification of critical parts of the promoter

When we induce mutations to make the promoter conform to the consensus sequence. The results show in general that the more a promoter looks like the consensus sequence, stronger the promoter will be (pg. 524)

What is lRNA, lncRNA, and sRNA? What are these used for?

lRNA= long RNA lncRNA= long, non-coding RNA sRNA= small RNA These help regulate mRNA (pg. 517)

Promoters recognized by RNA pol III

pg. 335