mmg2040 chapter 11 - transcription of genes

Define: cistron , ORF, promoter, terminator, activator, transcription factor, inducer, operator, repressor, enhancer, antisense/template strand, sense/coding strand

cistron: Segment of DNA (or RNA) that encodes a single polypeptide chain

ORF: Sequence can be translated (at least in theory) to give a protein

promoter: Region of DNA in front of a gene that binds RNA polymerase and so promotes gene expression

terminator: DNA sequence at end of a gene that tells RNA polymerase to stop transcribing

activator: Protein that switches a gene on

transcription factor: Protein that regulates gene expression by binding to DNA and/or to RNA polymerase

inducer: Small signal molecule that binds to a regulatory protein and causes a gene to be switched on

operator: Site on DNA to which a repressor protein binds

repressor: Regulatory protein that prevents a gene from being transcribed

enhancer: Regulatory sequence that binds transcription factors but that is outside, and often far away from, the promoter

antisense/template strand: the strand read by RNA pol to form new RNA molecule, complementary to the coding sequence

sense/coding strand: strand containing exact same sequence as the mRNA, complementary to the template strand

Name the two special sequences in a bacterial promoter and the two major
components of bacterial RNA polymerase

sequences in promoter:

• -10 region (Pribnow box) - TATAAT

• -35 region - TTGACA

Bacterial RNA pol consists of:

1) core enzyme (alpha, beta, etc.)

2) sigma unit for promoter binding

Describe how rifampicin inhibits the RNA polymerase of bacteria

Prokaryotic RNA polymerase is inhibited by the well-known antibiotic rifampi-
cin.

• This binds to the channel for DNA and RNA within the β-subunit of the
  enzyme

• Although rifampicin is not actually bound at the active site, it physically blocks elongation of the growing RNA chain

• Most rifampicin resistant mutations result from single changes in the amino acid sequence of the β-subunit that decrease rifampicin binding

Describe how termination of transcription occurs in bacteria

there is a special terminator sequence at the end. The terminator is in the template strand of DNA and consists of two inverted repeats separated by half a dozen bases and followed by a run of adenines (A’s)

• Once the RNA polymerase reaches the stem and loop, it pauses. Long RNA
  molecules contain many possible hairpin structures that cause RNA polymerase to slow down or stop briefly, depending on the size of the hairpin

• a string of U’s paired with a string of A’s in the
  template strand of DNA is a very weak structure, and the RNA and DNA fall apart

• The RNA polymerase “stutters” (due to steric hindrance) and the precise location of termination may vary slightly between different molecules of the same mRNA. Once the DNA and RNA have separated at the terminator structure, the RNA polymerase falls off and departs to find another gene

1) rho-dependent

• “intrinsic,” terminators do not need Rho or any other factor to cause termination

2) rho-independent

• Rho-dependent terminators are relatively frequent in bacteriophages

• The RNA sequence for Rho binding (rut sequence) is poorly defined but is high in cytosines and low in guanines

• Rho can only bind to the growing mRNA chain once the RNA polymerase has synthesized the rut recognition regions and moved on

• After binding to all six rut sites, Rho changes conformation to form a closed doughnut structure

• When Rho binds to its recognition site it causes allosteric changes in the catalytic subunits of the RNA polymerase that result in termination. Rho then unwinds the DNA/
  RNA helix in the transcription bubble and separates the two strands, leading to
  disassembly

Describe transcriptional regulation of maltose and lactose operons in bacteria

maltose: positive regulation - control by an activator that promotes gene expression

When there is maltose in this system, maltose binds with MalT to bind to sequence, gene is “switched” on as an activator

• activator: MalT

• inducer: Maltose

• depends on the whether there is maltose available or not

lactose: negative regulation - regulatory mode in which a repressor keeps a gene switched off unitl it is removed

The gene is not on until something happens as the protein is bound to the sequence already. lactose binds to laci and it leaves the region, this will allow transcription of the gene. The gene is switched on

• operator: laci

• inducer: lactose

• depends on whether lactose is available or not

Describe the function of the three different RNA polymerases in eukaryotes

• RNA polymerase I - Eukaryotic RNA polymerase that transcribes the genes for the large ribosomal RNAs

• RNA polymerase II - Eukaryotic RNA polymerase that transcribes the genes encoding proteins (the workhorse of the cell)

• RNA polymerase III - Eukaryotic RNA polymerase that transcribes the genes for 5S rRNA and tRNA

List the three major regions of an RNA Pol II promoter

• TFIID - Includes TBP – binds TATA box

• TFIIA - Binds upstream of TATA box; required for binding of RNA Pol II to promoter

• TFIIB - Binds downstream of TATA box; required for binding of RNA Pol II to promoter

• TFIIF - Accompanies RNA Pol II as it binds to promoter

Describe the importance of the TATA box

Binding site for TBP that guides RNA polymerase to promoter —> is needed for binding of RNA polymerases I and III. TBP is unusual in binding in the minor groove of DNA. (Almost all DNA-binding proteins bind in the major groove.) On both sides of the TATA box are G/C-rich regions

Describe events that help RNA Pol II to bind to a promoter and begin transcription

RNA polymerase II (Pol II) binds to promoters by assembling a Pre-Initiation Complex (PIC), causing DNA bending.

• Before RNA polymerase II can proceed, other factors must bind.

• TFIIH phosphorylates the tail of RNA polymerase III by recognizing the TATA box

• The tail changes position with respect to the body of RNA polymerase II

• The other factors leave and TFIIH helicase activity opens the DNA, and its kinase activity phosphorylates Pol II enabling transcription initiation

• RNA polymerase moves along the DNA and begins the process of transcription