Cell Biology Transcription

Describe the 3D structure of RNA.

*RNA is SINGLE-STRANDED; the base- pairing that occurs in RNA is all through regions of SELF-COMPLEMENTARITY. *Like DNA, RNA CANNOT adopt the B-form helix because the additional 2' hydroxyl interferes with the arrangement of the sugars in the phosphate backbone. Although, it can be found in the A-form and does base pair to form complex secondary and tertiary structures. *RNA can fold into MANY SHAPES and take on MANY FUNCTIONAL ROLES.

The sequences required for splicing are located where?

-5' junction, 3' junction, internal to the intron.

What do introns and alternative splicing allow for?

-INCREASES coding potential of genome +23,000 protein coding genes +Each gene has potential to form multiple proteins. -DOMAINS can be MIXED and MATCHED -Allows EVOLUTION of genes and proteins by removing, adding, or replacing entire modules.

T/F: A 5' cap and polyA tail is added to prokaryotic messages

FALSE

T/F All genes encode a protein.

FALSE *Some genes DO NOT encode polypeptides but encode structural or regulatory RNAs.

Where does eukaryotic translation occur?

In the cytoplasm, with RIBOSOMES!

What is the name of the factor in prokaryotic polymerase that recognizes the promoter?

Sigma Factor

T/F: TBP contains a polyglutamine (polyQ) repeat region.

TRUE

T/F: Only fully processed mRNAs are exported from nuclear pores.

TRUE *Processed mRNA is tagged with proteins marking them ready for export

How is the direction of transcription determined?

The direction of transcription is determined by orientation of the asymmetrical promoter sequence.

List 2 differences in the CHEMICAL makeup of DNA vs. RNA a. b.

a. Sugar Differentiation *Ribonucleotide uses a RIBOSE sugar; HYDROXYL group attached to the 2' carbon makes RNA MORE reactive. *Deoxyribonucleotide uses a DEOXYribose sugar; rather than a hydroxyl group, the 2' carbon is attached only to a HYDROGEN, making DNA LESS reactive. b. Base Pairing *RNA: URACIL pairs with adenine *DNA: THYMINE pairs with adenine

Transcription is the copy of __________ into ______________.

1) DNA 2) RNA

What defects in splicing can lead to disease? Name 3.

1) Factor VIII gene: hemophilia 2) CFTR gene: cystic fibrosis 3) Oncogenes: cancer

Name the 3 types of eukaryotic RNA polymerases and what genes they associate with.

1) RNA Polymerase I: most rRNA genes. 2) RNA Polymerase II: protein coding genes, miRNA genes, plus genes for some small RNAs (e.g. those in spliceosomes). 3) RNA Polymerase III: tRNA genes, 5S rRNA genes, genes for many other small RNAs.

Generalize transcription and the role of RNA polymerase.

1) RNA pol forms complex to DNA (with the aid of transcription factors, sigma factors, promoters, etc) and opens it up (forming TRANSCRIPTION BUBBLE). 2) RNA Pol moves stepwise along DNA (via the oxidation of ATP/GTP); unwinding DNA in front of it (modifying tension with topoisomerases), and adding nucleotides one at a time (RNA growing in 5'->3' direction); more error-prone than DNA replication. 3) Newly made RNA leaves at exit channel 4) RNA pol catalyzes formation of phosphodiester bonds, rewinding DNA.

What are some exceptions to the central dogma as it was first described?

1) Retroviruses -Viruses that use REVERSE TRANSCRIPTION; once inside the host cell's cytoplasm, these viruses use an enzyme called reverse transcriptase to produce DNA from its RNA genome. Violating the flow of the central dogma, allowing information to flow from RNA->DNA, then, following the model as expected; ex. HIV. *(RNA->DNA)->RNA->Protein 2) Prions -An INFECTED PARTICLE made up of MISFOLDED PRION PROTEINS; Violation of the central dogma because proteins are directly being SYNTHESIZED by other proteins (via CONFORMATION CHANGES). *Protein->Protein. 3) Primitive viruses -Some viruses are so primitive, that they synthesize proteins directly from RNA; not being able to produce DNA. *RNA->Protein.

List 5 types of RNA and what each type does. Also, what are snRNPs?

1) mRNAs: CODES for protein 2) rRNAs: FORM the CORE of the RIBOSOMES and CATALYZES protein synthesis 3) miRNA: REGULATE gene expression 4) tRNAs: serve as ADAPTERS between mRNA and AMINO ACIDS during protein synthesis 5) other small RNA: used in RNA SPLICING, TELOMERE MAINTENANCE, and many other processes. *snRNPs (snurps): RNA protein complexes that allow SLICING.

How is eukaryotic mRNA processed? What does the processing do for the mRNA? Be able to label the different regions of the mRNA.

Eukaryotic mRNA is PROCESSED before leaving the nucleus (during transcription); -the processing factors located on poly-adenylation tail -type of processing is specific to RNA type -mRNA is CAPPED at 5' end, SPLICED, and a poly-A tail is added to the end (3' cleavage). The processing of mRNA does the following; -Increase STABILITY of the mRNA molecule -Aid in nuclear EXPORT -IDENTIFY the molecule as mRNA -VERIFY lack of mRNA DEGREDATION prior to translation

T/F: All eukaryotic genes contain introns.

FALSE MOST eukaryotic genes contain introns; there are exceptions.

T/F: There are 3 bacterial RNA Polymerases.

FALSE There are 3 EUKARYOTIC RNA polymerases.

What is the splicing machinery composed of?

Each spliceosome is composed of five small NUCLEAR RNAs (snRNA), and a range of associated PROTEIN FACTORS. When these small RNA are combined with the protein factors, they make an RNA-PROTEIN COMPLEX called snRNP, or snurp. SMALL RNA + PROTEIN FACTORS = snRNP (snurp)

What does RNA Pol do to the DNA prior to transcription initiation?

In both eukaryotes and prokaryotes, RNA polymerase attaches to the DNA strand and forms a loose closed complex. Upon initiation of the breaking of H bonds, the RNA polymerase forms a tighter complex to the RNA to open up the DNA helix, separating the coding strand from the template strand (strand to use for RNA synthesis). *Topoisomerases are used to relieve tension from supercoiling in the DNA strand.

Are introns most characteristic of prokaryotic RNA, eukaryotic RNA, or both?

Introns are most characteristic of EUKARYOTIC RNA. Introns are noncoding regions of genes that tend to increase with complexity. Since eukaryotic organisms are MORE complex than prokaryotic organisms, introns are likely to be more plentiful in the eukaryotic genome. Remember: EXons = EXpressed sequences INTrons = INTervening sequences *Introns are larger than exons and most eukaryotic genes contain them; however, eukaryotic genes contain different amounts of exons and introns.

What must happen to Pol II before it can be released to begin transcription? How is this accomplished?

PRIOR to release for transcription, Pol II must be POSITIONED (initiation). TFIID (with TBP) binds to TATA box (and promoter) and distorts (BENDS) DNA. This TF is followed by the binding of TFIIB, which recognizes other promoter sequences (also helping to POSITION pol II). Together, with the help of other TF's, an initiation complex is formed; after phosphorylation of pol II CTD tail by KINASE subunit in TFIIH, transcription elongation begins. *NOTE: ORDER of ASSEMBLY of TF is PROMOTER-SPECIFIC!!!

Which basal TF phosphorylates pol II CTD?

PRIOR to release for transcription, the pol II tail must be PHOSPHORYLATED. This is accomplished by the KINASE subunit in TFIIH. Most general TFs are then released, free to initiate another round of transcription.

How quickly does RNA polymerase transcribe a typical gene?

RNA Pol can transcribe a gene in about 1 MINUTE! Many RNA transcripts can be made from one gene simultaneously.

Describe the differences and similarities between eukaryotic and prokaryotic transcription. In which cellular compartment does transcription occur in each?

RNA Polymerase -Prokaryotes: only produce 1 TYPE of RNA -Eukaryotes: produce 3 DIFFERENT TYPES of RNA Gene Regulation -Prokaryotes: more simplistic -Eukaryotes: more COMPLEX due to the chromatin structure and more regulatory sequences. Sigma Factor -Prokaryotes: have sigma factor attached to core enzyme. -Eukaryotes: require general TRANSCRIPTION FACTORS for transcription, instead of sigma factor. *In Prokaryotes, transcription occurs in the cytoplasm; TRANSCRIPTION and TRANSLATION occur SIMILTANEOUSLY! *In Eukaryotes, transcription occurs in the nucleus first; whereas, translation occurs via the ribosomes in the cytoplasm.

Which eukaryotic polymerase transcribes protein coding genes?

RNA polymerase II

Who won the Nobel Prize for studies on RNA Pol II structure?

Roger D. KORNBERG

Can RNA have enzymatic or structural function? Describe.

Yes, the single-stranded RNA can fold into SPECIFIC STRUCTURES, allowing it to have STRUCTURAL or CATALYTIC functions. ex. RIBOZYMES are RNA MOLECULES that are capable of CATALYZING SPECIFIC biochemical reactions including the cleavage or ligation of DNA, RNA, or peptide bond formation.

How is mRNA lifespan regulated and why is this important?

mRNA lifespan depends on RNA SEQUENCES in 3' untranslated region (UTR); it is another level of CONTROL for AMOUNT of GENE PRODUCT. However, mutations in 3' UTR of specific genes can prolong mRNA lifetime and lead to disease; -3' UTR mutations in CYTOKINES can lead to CHRONIC INFLAMMATION -3' UTR mutations in ONCOGENES can lead to CANCER

Can gene expression regulation occur at the transcriptional level, translational level, or both?

BOTH! *Eukaryotic gene expression is controlled at the levels of epigenetics, transcription, post-transcription, translation, and post-translation.

What must happen to eukaryotic RNA transcripts before translation?

Before translation, eukaryotic transcripts must be CAPPED, SPLICED, EJC (exon junction complex) is added when splicing is complete, and 3' CLEAVED/POLYADENYLATED.

What happens to the above factor after transcription initiation?

Following transcription initiation, the sigma factor is ejected from the RNA polymerase holoenzyme. After the RNA pol finishes synthesizing mRNA, the sigma factor binds to it once again.

What do the general transcription factors do? (3 main things)

General transcription factors inititate transcription by: 1) Positioning Pol II 2) Pulling apart the double helix 3) Launching Pol II to allow initiation of transcription

What is the structure that forms during the splicing process?

Lariat Formation

How does the splicing machinery know where to splice?

Splicing machinery (SPLICEOSOME) relies mostly on RNA and is guided by SPLICING SEQUENCES to cut out INTRON. -Branch point ADENINE in intron ATTACKS 5' SPLICE SITE, CUTTING sugar-phosphate backbone -5' END of cut intron COVALENTLY linked to 2'OH of ADENINE -FREE 3'-OH end of EXON reacts with start of NEXT EXON to JOIN them together -A LARIAT forms out of the intronic sequence

T/F: Alternative splicing can occur during cancer development.

TRUE

T/F: The Pol II CTD must be phosphorylated to be released and begin transcription elongation.

TRUE *CTD = C-terminal domain

T/F: Eukaryotic RNA must be fully transcribed before mRNA processing begins.

TRUE Processing occurs as RNA is transcribed.

Briefly describe the central dogma.

The CENTRAL DOGMA describes the flow of genetic information from DNA to RNA to protein; phrase coined by Francis Crick. 1) DNA is REPLICATED, REPAIRED, and RECOMBINATION occurs. 2) RNA SYNTHESIS (TRANSCRIPTION) 3) PROTEIN SYNTHESIS (TRANSLATION)

How does prokaryotic polymerase know where to start and stop?

The RNA polymerase holoenzyme binds loosely to the DNA strand, sliding along the strand until the SIGMA FACTOR recognizes the promoter sequence. Upon recognition, the RNA polymerase forms a tight complex with the DNA, opening up the helix (forming an open complex; transcription bubble) and synthesizing RNA using the template strand. The sigma factor is released as the elongation of the RNA chain ensues until the RNA polymerase reaches a termination sequence or signal (Rho-dependent or Rho-independent).

Which protein binds the TATA box? Which protein complex is this a part of? What does binding of this factor do to the DNA? Why is this important for transcription?

The TFIID locates the TATA box and binds to the pol II promoter, 25 bp UPSTREAM of the transcription start site. The TATA BINDING PROTEIN (TBP) is part of TFIID complex and BINDS to the TATA box, which causes the DNA to distort. The BENDING of the DNA (caused by the TFIID complex) is important for transcription because it creates a LANDMARK for other TF's.

What is a prion and why is this an exception to the central dogma?

This small infectious particle is a DISEASE-CAUSING form of a protein called CELLULAR PRION PROTEIN (PrP^c). *PrP^c (high ALPHA-HELICAL content) is mainly found on the SURFACE of CELLS in the CNS, but it is also located in other bodily tissues. Once propagated by PrP^sc (high BETA-SHEET content, aggregates in neural tissue, may act as a TEMPLATE for misfolding), PrP^c is transmitted into a misfolded protein state, causing the affected proteins to (progressively) CHANGE CONFORMATIONS; causes transmissible spongiform encephalopathies in mammals, which is why the affected brains appear "spongy" and with holes. *Cows: bovine spongiform encephalopathy, or "mad cow disease" *Humans: Creutzfeldt-Jakob Disease (CJD). ***It is an exception to the central dogma because proteins are directly REPLICATING themselves by making CONFORMATION CHANGES to other proteins. Protein--->Protein