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RNA vs DNA Structure

-one difference concerns types of bases found within the two species

-use three of the same bases (A, G, and C)

-fourth base is different T in DNA but U in RNA

-second difference concerns type of sugar that is found within nucleotide.

-DNA nucleotides contain deoxyribose sugar while RNA nucleotides contain ribose sugar

-RNA consists of a single polynucleotide strand that can fold on itself and bases can form complementary pairs (A:U and C:G)


Transcription Main Steps

-(1) initiation of transcription includes recruitment of RNA polymerase to core promoter, the localized melting of double helix and synthesis of up to the first 50 bases of the mRNA transcript

-(2) elongation step involves RNA polymerase continuing along DNA template and transcribing all exonic and intronic sequences into the mRNA

-(3) last step is regulated process of disengaging the RNA polymerase from DNA template (termination)

-mRNA must then be protected at both 5' and 3' ends from activity of nucleases

-A methylated guanine nucleotide (the 5' cap) is added to the 5' end.

-dozens to hundreds of adenine nucleotides (poly A tail) is added to 3' end of transcript


Transcription Elongation Through Nucleosomes

-RNA Poll II transcribes region of DNA into mRNA it must displace all proteins bound to double helix

-nucleosome difficult to displace since it is large complex and binding to DNA is very stable

-some studies suggest entire nucleosome disassembles ahead of advancing RNA Pol II and is translocated to region just behind RNA Pol II by chaperone proteins

-other data indicates in some cases only a subset of nucleosome proteins is displaced and reloaded onto nucleosome after RNA Pol II passes

-last mechanism suggests that hyperacetylation of nucleosomes ahead of RNA Pol II is sufficient to displace entire octamer. Chaperone then moves octamer to region of naked DNA behind RNA Pol II where it is methylated and de-acetylated

-thought that one general transcription factor TFIIS mediated the displacement of entire nucleosome and H2a-H2B dimer


Protecting 5' End of mRNA

-eukaryotic mRNAs need protection from degradation by normal cellular enzymes

-first modification occurs at 5' end

-to all mRNAs guanine residue is added by guanylyltransferase enzyme

-next addition of methyl group onto guanine nucleotide by methyltransferase

-if enzyme adds group to guanine DNA nucleotide by mistake this needs be to repaired by DNA damage repair pathways


Purposes of Methylated 5' End

-helps protect 5' end from degradation

-serves as signal for ribosome to bind to 5' end of transcript and initiate translation

-during translation 5' cap is bound by proteins that can also interact with factors that are bound to 3' end of mRNA

-creates circular more stable piece of RNA


Processing and Protecting 3' End of mRNA

-in eukaryotes, transcription termination signal encoded with a sequence specific element at 3' end of each gene

-upon reading this sequence of bases RNA polymerase will disengage from DNA strand

-CstF (normally attached to RNA polymerase) binds to sequence element within mRNA transcript and cleaves it into 2 pieces

-main body of primary transcript will be further modified at 3' end while shorter end piece is degraded

-second protein called CPSF (also bound to RNA polymerase) binds to sequence element within mRNA transcript called poly-A signal sequence

-once bound to mRNA, CPSF recruits PAP (Poly-A polymerase)

-PAP binds to very 3' end of RNA and adds string of adenine nucleotides to end of mRNA transcript. The average length of added adenine residues is 200

-presence of poly-A tail acts as buffer while nucleases are digesting the end of transcript

-length of poly-A tail directly proportional half-life of mRNA transcript

-Poly-A Binding Protein (PABP) binds to and stabilizes poly-A tail

-also interacts with proteins that bind to 5' cap which allows mRNA transcript to adopt circular conformation


Structure/Transcription of Eukaryotic Gene

-transcription of any eukaryotic gene is controlled by core promoter and enhancer elements

-all core promoters bound by set of common general (basal) transcription factors

-serve to recruit RNA polymerase and together are essential for initiating transcription

-decision of where and when to activate transcription is made at enhancer element

-developmentally regulated transcription factors will bind to enhancer element and exert temporal and spatial control of transcription

-proteins that bind to enhancer element can be transcriptional activators or repressors

-during transcription enhancer and core promoter not transcribed

-elements that are transcribed include all exons and introns

-primary mRNA transcript will be spliced to yield a mature mRNA species that contains only exons (thus lacking intronic sequences)

-first and last exons will contain sequences that are transcribed but not translated



-in both prokaryotes and eukaryotes the first base that is transcribed within a gene is called the transcriptional start site

-given the designation +1 and is often referred to as the +1 site

-any base located upstream or 5` of the transcriptional start site is given a negative number while any base that is located downstream or 3` of the transcriptional start site is given a positive number


Structure of Prokaryotic Core Promoters

-all bacterial core promoters contain a 6 bp sequence

-specific element starting 10bp upstream (-10 element) of the transcriptional start site

-in addition many core promoters will contain a second 6pb sequence-specific element starting 35bp upstream (-35 element) of the transcriptional start site

-small set of core promoters will containeither additional sequence of sequence-specific element called upstream of promoter (UP) element or a larger element

-all these elements located upstream of transcriptional start site and assigned negative numbers


Structure of Eukaryotic Core Promoters

-are organized quite differently than prokaryotic

-contain many more sequence-specific elements than their prokaryotic counterparts

-these elements can also be found straddling the transcriptional start site or can be located either upstream or downstream of this base


Sigma Factor (What?)

-in prokaryotes protein called Sigma Factor recognizes and binds to the -10, -10 extension, and -35 elements

-multi-domain protein

-protein dissections have identified regions of the protein that bind to individual core promoter elements


How Sigma Factor Works

-ex: 2.4 domain of the Sigma Factor recognizes and binds to the -10 element, the 3.0 domain binds to the -10 extension and the 4.2 domain binds to the -35 element

-only core promoter element that is not contacted by the Sigma factor is the UP-element

-serves as general transcription factor

-bound to core promoter element of all genes and recruits RNA polymerase to promoter

-RNA polymerase itself is multi-subunit protein -one of alpha subunits is responsible for contacting Sigma Factor

-Sigma Factor is also responsible for local "melting" of DNA helix in preparation for RNA polymerase to begin transcribing template strand into mRNA


General Transcription Factors and Eukaryotic Core Promoter

-contains several sequence-specific elements

-all promoters will contain Inr and BRE elements

-about 50% of core promoters will have a four base (TATA) recognition element

-remaining 50% of core promoters that lack a TATA box, they contain one of two types of sequence specific elements: downstream promoter element (DPE) or the downstream core element (DCE)

-these five different core promoter elements are bound by two general transcription factor complexes: TFIIB and TFIID. -

the Inr, TATA, DPE and DCE elements are bound by TFIID while the BRE element is bound by TFIIB



-a multi-subunit protein that contains TATA Binding Protein (TBP)

-first general transcription factor to bind to the core promoter

-followed by TFIIA which aids TBP binding to the TATA box and TFIIB which binds to the transcription factor IIB recognition element (BRE)

-this core set of general transcription factors is sufficient to recruit RNA polymerase to the Inr element of the core promoter

-TFIIF is recruited along with RNA polymerase and is required to stabilize interactions with TFIIB and TFIID