CHEM 114C Midterm 2

core enzyme

• the bacterial core RNA polymerase

• carries RNA synthesis

• alpha, beta, beta’, sigma, omega

sigma subunit

• helps find promoters and participates in the initiation of RNA synthesis

• activates. in the beginning of synthesis

• released after synthesizing 10-15 nucleotides

holoenzyme

• formed when the sigma subunit joins the core enzyme

• complete enzyme

-10 sequence

• 6-base-pair-long sequence located about 10 nucleotides upstream from the start site (to the left)

• sequence that helps with RNA polymerase to bind and start translation process

-35 sequence

• 6 base pair long sequence located about 35 nucleotides upstream from the start site

• sequence that helps with RNA polymerase to bind and start translation process

core promoter

the -10 sequence and -35 sequence together

bacterial promoter sequences

• identifying which letters are used the most for each sequence

• helps to identify the important sequences that carry out a function

How do we know that these sequences are important?

1. high conservation

2. binding by RNA holoenzyme which shields that DNE from enzymes that will attack DNA: DNAse I or DMS footprinting

3. Functional tests: mutation affects the rate of transcription (ex. Firefly Lucifrase & GFP)

DNAse I Footprinting

a method that compares the two 32P end-labeled fragments, one with specific binding protein and the other with no protein, and through gel electrophoresis, it compares the gels and the gap between the bands is a footpring that tells the sequence to which the protein is binding

Dimethylsulfate (DMS) Footprinting

• an experiment that is carried out to identify a sequence that prefers to bind to a particular sequence

• modifies the DNA and adds methyl group

• chemical is preferred (DMS is preferred) for the footprint then the enzyme because the chemical is so small it can get inside into the solvent accessible sites of the enzyme while enzymes are too big to do that

• if chemical is used, it gives a clear band compared to an enzyme

Steps for DMS Footprinting

1. End-label DNA fragment

2. Bind protein

3. Treat with dimethylsulfate, which methylates guanines. *Guanines that are bound by proteins cannot be methylated. But single-stranded guanines are more prone to methylation.

4. Remove protein

5. Cleave DNA by depurinating. the methylated bases (piperidine)

6. Separate DNA fragments on denaturing (8M urea) gel

Bacterial Core Promoter Consensus Sequence

• 2 common motifs are present on the upstream side of the transcription site

• Known as -10 sequence and the -35 sequence

• optimal distance between these two sequences is 17 nucleotides

Strong Promoters

• have sequence that correspond closely to the consensus sequence

• initiate every 2 seconds in E. coli

Weak Promoters

• have sequence that deviates from the consensus sequence

• cannot recruit RNA polymerase

• initiate every 10 minutes in E. coli

Upstream Element

• about 40 to 60 nucleotides of the core promoter are sequences that can enhance transcription

• interact with the alpha-subunit of RNA polymerase

RNA polymerase holoenzyme Complex

• sigma subunit contacts the -10 to -35 sequences and recognizes them

• sigma subunit is generally released when the nascent RNA chain reaches 9 or 10 nucleotides in length

• just switching the sigma subunit changes the gene expression (core enzyme remains the same) and can regulate transcription

• E. coli has 7 distinct sigma factors

Alternative Promoter Sequences

• sigma 70 = recognize the consensus promoter (-10 and -35 sequences)

• sigma 32 = recognize the promoters of heat-shock genes which helps to stabilize the cellular proteins (ex. help with some folding)

• sigma 54 - recognize promoters of nitrogen-starvation genes

• streptomyces coelicolor encodes more than 60 sigma factors

DNA unwinding by RNA polymerase

• transition from closed promoter complex (DNA duplex) to open promoter complex (RNA polymerase binds and separates the DNA strands) requires the unwinding of about 17 base pairs of DNA

• free energy necessary for it comes from additional interactions between the single stranded DNA and the enzyme

• -35 element remains double stranded & -10 element is unwound

Transcription Bubble

• RNA polymerase sometimes fall off after adding less than 10 nucleotides

• the newly synthesized RNA forms a RNA-DNA helix about 8 base pairs long

• the core enzyme contains a binding site for the coding strand

• starts from 3’ end which is where the enzyme is added to add new NTP

Rho-independent termination

• the secondary structure of RNA that was newly transcribed is going to catalyze its own termination

• does not need any protein

• RNA transcript is self-complementary forming a hairpin structure

• RNA polymerase pauses after the hairpin is formed

• RNA-DNA hybrid helix is unstable because of the weaker rU-dA base pairs → allows the RNA transcript to dissociate from the DNA template

Hairpin

works for rho-independent when the RNA reaches the U’s, the GC rich sequence is going to snap into place and slows down the RNA polymerase due to a very stable secondary structure

Rho protein helps to terminate the transcription of some genes

• rho protein binds to the newly transcribed RNA (5’ end) and travels along the RNA and then it contacts the RNA polymerase

• when contacting the RNA polymerase, since rho protein is a powerful helicase, it yanks/pulls the RNA out of the active site of the RNA polymerase and cause termination

• promotes about 20% of termination events

• hydrolyzes ATP in the presence of single-stranded RNA that travels on that single stranded RNA

Rho

• consists of 8 subunits that come together

• detects additional termination signals that are not recognized by RNA polymerase alone

• homologous to hexameric helicases

Mechanism for rho-dependent termination

• rho hydrolyzes ATP in the presence of ssRNA but not in the presence of DNA or duplex RNA

• rho binds to the RNA transcript and chases the RNA polymerase, which breaks the RNA-DNA hybrid at the transcription bubble and pulls RNA out of the active site

Rho-dependent Transcription Termination

• if rho is present at the start of synthesis and add along with RNA polymerase, it terminates transcription very quickly

• the experiment illustrates how rho actually has to start travel on the RNA & find the appropriate termination sequence

Sedimentation coefficient

characterizes its sedimentation during centrifugation. defined as the ratio of a particle’s sedimentation velocity to the applied acceleration causing the sedimentation

Mechanism for the Termination of Transcription by Rho

1. RNA polymerase transcribes DNA, which is the sequence is rich in cytosine residues

2. Rho attaches to recognition site on RNA

3. Rho moves along RNA, following RNA

4. RNA polymerase pauses at terminator

5. Rho unwinds DNA-RNA hybrid

6. Termination

Riboswitches

• regulatory RNAs located in the upstream untranslated region that bind small molecules to regulate gene expression

• found across the eubacteria domain

• can regulate transcription or translation

  • transcription termination → can turn off the expression of that particular mRNA

  • translation initiation

  • mRNA processing

• do not require any intermediary sensor molecules (proteins or RNAs)

Riboswitch function

1. Transcription termination sequence is sequestered by aptamer structure → transcription termination sequence is free, gene expression is blocked

   • regulates transcription

   • regulates whether mRNA synthesizes or not

   • does so by forming a stem loop/hairpin to terminate transcription

2. Structure of RNA aptamer sequesters the ribosome binding site and inhibits translation → RBS is free and translation may ensue

   • regulate translation

   • under the presence of ligand which it can fold into a structure & prevents mRNA from forming to the ribosome

3. a variety of different sequences & structures of Riboswitches that recognizes different types of ligands

• look at slides 20-21 in lecture 10

Sequester

• masked and it is not available

• don’t get translation; no gene expression

FMN Riboswitch

• FMN = flavin mononucleotide

• control of riboflavin biosynthesis in Bacillus subtilis

• mRNA can form special structures that can bind small molecules

• the U sequence allows the hairpin structure to fall off from the active site of the RNA polymerase

• @high FMN concentration: FMN binds to the mRNA that forms a terminator structure and prevents the production of functional mRNA → turns off the formation of hairpin

• @low FMN concentration: an alternate base pair form which prevents the formation of hairpin that allows the RNA polymerase to synthesize the full length mRNA

Recognition of FMN by its riboswitch

• FMN forms the Watson & Cricks base pair with the riboswitch that allows the riboswitch to discriminate other molecules binding to riboswitch

• Riboswitch can form very precise interactions that allows to discriminate between a variety of different ligands

Mapping Experiment

• allowed the scientists to determine what the overall shape is of the riboswitch

• venom (V1) = double stranded RNA

• T2 = single stranded RNA

Rifampicin

• widely used antibiotic used treat disease (ex. tuberculosis)

• semisynthetic derivative of rifamycins which are produced by a strain of streptomyces

Structure of Rifampicin

• grows in a competitive environment

• consumes a limited supply of nutrients so they can kill the bacteria surrounding them to form the complex structure to be much more successful in the technological niche

Rifampicin Blocks the initiation of RNA synthesis

• Rifampicin binds to a pocket in the RNA polymerase that is normally occupied by the newly formed RNA-DNA hybrid

  • Rifampicin only binds to bacterial RNA polymerase

• Rifampicin blocks elongation after only two or three nucleotides have been added

  • can only inhibit the early states of transcription (about 12A away from the active site)

  • if it b

Actinomycin-DNA complex structure

• produced by streptomyces

• binds tightly and specifically to double stranded DNA and prevents it from being an effective template for RNA synthesis

• Actinomycin D is not acting on the RNA polymerase

• prevents transcription by RNA polymerase

• @high concentration: can inhibit DNA replication/any process that requires strand separation

• @low concetration: inhibits transcription without significantly affecting DNA replication or protein synthesis

• phenoxazone ring slips in between base pairs in DNA (intercalation)

  • the two hooks will hold the 2 strands together & prevents the separation of DNA strands

• inhibits RNA synthesis in both prokaryotic and eukaryotic cells

  • cannot be used to treat bacterial infection

• has the ability to inhibit the growth of rapidly growing cells makes it useful for the treatment of some cancers

Primary transcript

• prokaryotes: mRNA undergoes little or no modification after synthesis

• tRNAs and rRNAs are generated by cleavage and other modifications of the nascent RNA chains

• 16S rRNA (part of small subunit), 23S and 5S rRNA (part of large ribosomal subunit)

• look at slide 7 in lecture 11 for the general organization of the ribosomal RNA operons

Ribonuclease P (RNase P)

• has an RNA component & a protein components

• generates the correct 5’ terminus of all tRNA molecules in E. coli

• RNA component carries out the trimming reaction that removes the 5’ end of all tRNAs to give the mature tRNA that participates in translation

  • result: 5’ phosphate & 3’ hydroxyl group

• highly conserved reaction

• every organism have RNaseP to carry out the reaction

• RNA component is the catalytic subunit

• found by Signey Altman

RNase P mechanism

1. RNase P binds to the metal ion

2. carries out phosphodiester cleavage reaction

• RNA molecule adopts a planar structure

  • all RNA helical parts are coaxly stacked

  • tRNA is coming from one side

Ribonuclease III (RNase III)

• excises 5S, 16S, and 23S rRNA precursors from the primary transcript by cleaving double-helical hairpin regions at specific sites

RNA modifications

• enzymatic modifications of the standard bases is common in tRNAs and rRNAs

• ribose unit are also modified

• generates diversity allowing greater structural and functional versatility

• hypothesis: modifications can change the stability of the RNA

• 2 types of modification:

  • Ribothymidylate: methylation from uracil to thymine

  • Pseudouridylate

Transcription in Eukaryotes

• transcriptional control

  • key driving force for differentiation

  • major mechanism used to regulate gene expression

• multicellular eukaryotes use differential transcriptional regulation to create different cell types

Transcription & Translation in Prokaryotes

• no nucleus; no separation of DNA & ribosomes

• as RNA polymerase synthesizes and transcribes mRNA, ribosomes attaches & begins translating at the same time → co-transcriptional translation

• bacteria only have 3 promoter elements (-10, -35, and UP elements)

• mRNA is unmodified in prokaryotes

Transcription & Translation in Eukaryotes

• DNA is located in nucleus & ribosomes are in cytoplasm

• transcription and translation are not coupled

• uses a variety of types of promoter elements, each identified by its own conserved sequence

  • these elements can be located upstream, downstream, and sometimes quite far from the start site

  • various combinations of these elements are used to initiate transcription

• very extensive RNA process destined to become mRNA

  • splicing out introns, 5’ CAP addition, 3’ Poly A tail addition

Enhancer

• a proximal distal elements

  • the other one is proximal proximal elements

• can bind to a lot of transcriptional factors

bacterial transcriptional machinery

RNA polymerase & some core proteins are bound

Data box

where RNA polymerase binds to begin transcription

CAP

• 7 methylguanine structure

• recognized by EIF4E (eukaryotic initiation factor)

EIF4E

a cap-binding protein that 4E binds to the 7-methylguanosine CAP that is important for the assembly of the translation initiation complex

3’ Poly A tail addition

a tail of poly-A that binds to poly-A binding protein which is important for translation