Chapter 26 - RNA Metabolism

Define Coding and Template strands.

1) DNA template strand - serves as template for RNA polymerase 2) DNA coding strand - the non-template strand; has the same sequence as the RNA transcript

Describe RNA polymerase III and IV.

1) RNA polyermase III makes tRNAs and some small RNA products -Plants appear to have RNA polymerase IV that is responsible for synthesis of small interfering RNAs

Describe the chemistry of transcription in E. coli. What bonds are formed? What cofactors are used?

For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound. (a) Catalytic mechanism of RNA synthesis by RNA polymerase. The reaction involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleoside triphosphates (NTPs) and to three Asp residues, which are highly conserved in the RNA polymerases of all species. One Mg2+ ion facilitates attack by the 3'-hydroxyl group on the α phosphate of the NTP; the other Mg2+ ion facilitates displacement of the pyrophosphate, and both metal ions stabilize the pentacovalent transition state.

Describe the processes of termination w/o Rho.

) ρ-Independent termination. RNA polymerase pauses at a variety of DNA sequences, some of which are terminators. One of two outcomes is then possible: the polymerase bypasses the site and continues on its way, or the complex undergoes a conformational change (isomerization). In the latter case, intramolecular pairing of complementary sequences in the newly formed RNA transcript may form a hairpin that disrupts the RNA-DNA hybrid or the interactions between RNA and polymerase, or both, resulting in isomerization. An A=U hybrid region at the 3' end of the new transcript is relatively unstable, and the RNA dissociates from the complex completely, leading to termination.

Describe splicing of Group I introns.

-3'-OH of free guanosine is used as nucleophile -Attacks phosphodiester bond between U and A at the end of the intron -Releases first portion of U-ending exon -The 3'-OH of the U-ending exon then attacks the 5'-end of the other piece of exon to rejoin the pieces

Describe the 5'-cap.

-7-methylguanosine links to 5' end via 5', 5'-triphosphate link -methyl group derived from s-adenosylmethionine -Protect RNA from nucleases -Forms a binding site for ribosome -Triphosphate link also prevent degredation by nucleases (at thhat link)

Can RNA polymerase be selectively inhibted?

-Actinomycin D and Acridine intercalate DNA and prevent transcription -Rifampicin binds to beta-subunit of bacterial RNA pols

How are tRNA and rRNA processes?

-Basses are modified in post-transcription reaction into: 1) Pseudouridine 2)Thiouridine 3)Dihydrouridine -rRNAs and tRNAs are cleaved from longer precursors

Describe Rho-independent.

-Chracterized by the 3 U bases near the 3' end of the transcript and palidromic sequence -Self complementary regions in the transcript for a hairpin before the 3' end -Makes RNA poly stall and disscoaite

Does only one DNA strand encode proteins? Provide an example of the answer.

-Coding information may be on either strand -This is seen in the adenovirus

Describe the processes of termination with Rho.

-Common CA-rich sequence called rut site (Rho utiliation element) -Rho protein is a helicase, binds to rut site -Rho protein proceeds until temrination site is reached

How are most Eukaryotic RNAs processed after synthesis?

-Elimination of introns; joining exons -Polyadenylation of the 3' end -Capping the 5' end

Describe elongation and termination of RNA polymerase.

-Elognation factors bound to RNA Pol II enahce processivity and coordinate post-translational modification -For terminal, pol II is dephosphroylated

What is alpha-amanitin?

-From mushrooms -Blocks Pol II and Pol II of predator -Does not block its own Pol II

Describe Helicase and Kinase activity in RNA polymerase.

-Helicase activity in TFIH unwinds DNA at the promoter -Kinase acitvity in TFIH phosphorylates the polymerase at the carboxy-terminal domain changing the conformation and enabling RNA Pol II to trasncribe

What initiates assemble of RNA polymerase at the promoter?

-Initiated by TATA-binding protein (TBP) with the promoter -TBP is mart of a multisubunit complex TFID

What are introns?

-Most genes in vertebrates -Exons usually <1000 bp in length -Introns 50-20, 000bp in length -Some genes have dozens of introns

How are poly(A) tails added?

-RNA Pol II sythesizes RNA beyodn the cleavage signal sequences -Clevage signal is bound by an endonuclease and a polyadenylate polymerase bound to CTD of RNA Pol -Endonuclease cleave RNA downstream to highly conserved AAUAA -Polyadenylate polymerase synthesis 80bp of A

How many different end product can a single gene yield?

-RNA can be edited -Clevage/polyadenylation patterns can vary yielding different mRNA -Immunoglobulin heavy chain gene: different degrees of polyadenylation and different cleavage sites -Calcitonin and calcitonin-gene related peptide, in rat thyroid and brain made from same mRNA

Where does RNA polymerase bind? Describe the growing end? What primers are used? What type of coiling occurs?

-RNA polymerase binds to seqeunce called promoter to being transcription (primer not required) -Growing end of the new RNA temporarily base-pairs with DNA template for ~8bp -DNA duplex unwinds, forming a bubble of a few bp -RNA pol generates positive supercoil ahead, later relived by topoisomerases

Describe the structure of RNA polymerase? what abilities does it have?

-RNA polymerase is a holoenzyme that has five core subunits of alpha, beta, betaprime, sigma and -RNA Pol lacks 3'->5' exonuclease activity so has high error rate -RNA binds promoter regions to initiate transcription

What proteins does mRNA transcription in Eukaryotes have?

-Relies on protein-protein contacts (many highly conserved TFs) -RNA Pol II which is a large complex of 12 subunits and has a carboxy-terminal domain of highly conserved repats

How are RNAs synthesized? How are they folded?

-Ribonucleic acids are synthesized in cells using DNA as a template in transcription -ssRNA molecules can fold into compact structures with specfic functions (i.e. ribozymes that at as catalysts)

What is an example of enzymatic acitivty?

-Some introns have the ability to carry out their own splicing ribozymes

How are splicesome introns removed.

-Spliceosome made up of snRNPs (small nuclear ribnuclear proteins) -snRNP RNA is snRNA (for small nuclear RNA ) -5 snRNAs known in eukaryotes (U1, U2, U4, U5, U6) -GU at 5'-end and AU at 3'-end usually makr sites of splicing in mRNA

What does processing of mRNA include?

-Splicing out introns and rejoining exons for a continous sequences -Adding 5'-cap -Adding a 3'-poly(A) tail -Degradation

Describe splicing of group II introns.

-The nucelophile is a 2'-OH of an A residue within the intron -After the first cleavage, the second (right-most) piece form a lariat-like intermediate with a 2'-5'-phosphodiester bind -Other features are similar to group I introns

Describe transcription in E. coli.

-The nucleoside triphosphates add to the 3' end of the growing RNA strand -The growing chain is complementary to the template strand in DNA -The synthesis is catalyzed by enzyme (RNA polymerase) -RNA polymerase covers about 35 bp-long segment of DNA

What does TFIIH have do with repair.

-Transcribed genes are more actively repaired than silent genes -May be partly explained that TFIIH also has role in nucleotide-excision repair (NER) -Genetic rapir diseases are associated with TFIIH defect

What is a major target for regulation? Why? How?

-Transcription is energy-intensive therefore logical to regulate gene production here -One mechanisms is regulation affinity for RAN polymerase for a promoter 1) Promoter sequence 2) Activator proteins 3) Repressor proteins

Describe the Transcription Bubble?

. For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound. (b) About 17 bp of DNA are unwound at any given time. RNA polymerase and the transcription bubble move from left to right along the DNA as shown, facilitating RNA synthesis. The DNA is unwound ahead and rewound behind as RNA is transcribed. As the DNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand is extruded.

Describe the 5 steps in RNA intitation and elongation.

. Initiation of transcription requires several steps generally divided into two phases, binding and initiation. In the binding phase, the initial interaction of the RNA polymerase with the promoter leads to formation of a closed complex, in which the promoter DNA is stably bound but not unwound. A 12 to 15 bp region of DNA—from within the -10 region to position +2 or +3—is then unwound to form an open complex. Additional intermediates (not shown) have been detected in the pathways leading to the closed and open complexes, along with several changes in protein conformation. The initiation phase encompasses transcription initiation and promoter clearance (steps 1 through 4 here). Once elongation commences, the subunit is released and it is replaced by the protein NusA. The polymerase leaves the promoter and becomes committed to elongation of the RNA (step 5). When transcription is complete, the RNA is released, the NusA protein dissociates, and the RNA polymerase dissociates from the DNA (step 6). Another σ subunit binds to the RNA polymerase and the process begins again.

Describe the mechanisms of the addition of a Poly(A) tail.

. Pol II synthesizes RNA beyond the segment of the transcript containing the cleavage signal sequences, including the highly conserved upstream sequence (5')AAUAAA. This cleavage signal sequence is bound by an enzyme complex that includes an endonuclease, a polyadenylate polymerase, and several other multisubunit proteins involved in sequence recognition, stimulation of cleavage, and regulation of the length of the poly(A) tail, all of which are tethered to the CTD. 1 The RNA is cleaved by the endonuclease at a point 10 to 30 nucleotides 3' to (downstream of) the sequence AAUAAA. 2 The polyadenylate polymerase synthesizes a poly(A) tail 80 to 250 nucleotides long, beginning at the cleavage site.

Describe the six subunits of RNA polymerase.

1 )The alpha subunits function in assembly and binding to UP (upstream promoter) elements 2) The beta subunit is the main catalytic subunit 3) the beta prime subunit is responsible for DNA binding 4) The sigma subunit direct enzyme to the promoter 5) The w subunit appear to protect the polymerase from denaturation (stability)

What are the 4 classes of introns?

1) Group I and II (self splicing) -Require no additional proteins or ATP -In nuclear, mitochondrial and chlorplast genomes 2) Spliceosomal introns are spliced by enormous complexes called splicesomes 3) tRNA introns are spliced by protein-based enzymes -Primary transcript cleaved by endonucleases -Exons are joined by ATP-dependent ligases

What are the several steps of transcription intitation and elongation?

1) RNA Pol binds to promoter and creates a closed complex (DNA is not unwound) 2) Open complex forms (region from ~-10 to ~+2 unwinds) 3) RNA Pol moves away from promoter (sigma subunit is replaces by protein NusA)

Describe RNA polymerase I and II.

1) RNA polymerase I synthesizes pre-ribosomal RNA 2) RNA polymerase II is responsible for synthesis of mRNA -Very fast -Specifically inhibited by mushroom toxin -can recognize thousands of promoters

What are the types of termination in E. Coli?

1) Rho-independent 2) Rho-dependent

What common features to promoters have?

1) Two consensus sequences at -10 (TATAAT) and -35 (TTGACA) for sigma subunit (a TF) binding 2) A-T-rich upstream promoter element between -40 and -60 binds the alpha subunit 3) A-T-rich sequences promote strand separation 4) These sequences promote RNA Pol binding and therefore affect gene expression level

Describe the 3 main RNAs.

1) mRNA: Encode the amino acid sequences of all the polypeptides found in the cell 2) tRNA: Match specific amino acids to triplet codons in mRNA during protein synthesis 3) rRNA: Constituens of ribosomes and act as ribozymes catalyzing formation of peptide bonds

What are miRNA?

Appear to regulate the expression of genes, possibly via binding to specific nucleotide sequences

Describe the splicing mechanism of Group I intron

Can be GMP, GDP, GTP

Describe the positive supercoils generated by RNA polymerases.

For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound. (c) Movement of an RNA polymerase along DNA tends to create positive supercoils (overwound DNA) ahead of the transcription bubble and negative supercoils (underwound DNA) behind it. The RNA polymerase is in close contact with the DNA ahead of the transcription bubble as well as with the separated DNA strands and the RNA within and immediately behind the bubble. A channel in the protein funnels new NTPs to the polymerase active site. The polymerase footprint encompasses about 35 bp of DNA during elongation.

Describe Rho-Dependent.

Rho protein binds RNA and triggers dissociation

What are some promoters recognized by eukaryotic RNA polyermase II.

The TATA (-30) box is the major assembly point for the proteins of the preinitiation complexes of Pol II. The DNA is unwound at the initiator sequence (Inr), and the transcription start site is usually within or very near this sequence. Many additional sequences serve as binding sites for a wide variety of proteins that affect the activity of Pol II. These sequences are important in regulating Pol II promoters and differ greatly in type and number, and in general the eukaryotic promoter is much more complex than suggested here . Many of the sequences are located within a few hundred base pairs of the TATA box on the 5' side; others may be thousands of base pairs away.

Describe coding organization in adenovirus?

The genetic information of the adenovirus genome (a conveniently simple example) is encoded by a double-stranded DNA molecule of 36,000 bp, both strands of which encode proteins. The information for most proteins is encoded by (that is, identical to) the top strand—by convention, the strand oriented 5' to 3' from left to right. The bottom strand acts as template for these transcripts. However, a few proteins are encoded by the bottom strand, which is transcribed in the opposite direction (and uses the top strand as template). Synthesis of mRNAs in adenovirus is actually much more complex than shown here. Many of the mRNAs derived using the upper strand as template are initially synthesized as a single, long transcript (25,000 nucleotides), which is then extensively processed to produce the separate mRNAs. Adenovirus causes upper respiratory tract infections in some vertebrates.

Describe Transcription at RNA II promoters

The sequential assembly of TBP, TFIIB, TFIIF plus Pol II, TFIIE, and TFIIH results in a closed complex. Within the complex, the DNA is unwound at the Inr region by the helicase activity of TFIIH and perhaps of TFIIE, creating an open complex. The carboxyl-terminal domain of the largest Pol II subunit is phosphorylated by TFIIH, and the polymerase then escapes the promoter and begins transcription. Elongation is accompanied by the release of many transcription factors and is also enhanced by elongation factors. After termination, Pol II is released, dephosphorylated, and recycled.

Why does RNA polymerase need helicase?

Topoisomerase allows RNA polymerase to remain straight while replicating to prevent it from getting tangled up in DNA

Describe U1 and U2 snRNPs.

U1 helps define the 5'-splice site -U2 binds near the 3'-end of the intron -The Us bind to create a spliceosome -ATP required for assembly but not cleavage -Come parts attached to CTD of RNA Pol II