transcription and regulation
explain the key role of TATA-box-binding protein (TBP) in assembling active TFII transcription complexes
https://www.quora.com/What-is-a-TATA-box TBP is a common sequence feature in RNA Pol II promoters TATA box (TATAAA) near base pair -30 and an initiator Inr (YYANTAYY) sequence near the RNA start site at +1 5' side-----(-30)----(+1)----3' side 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 and are located within a few hundred to a few thousand base pairs of the TATA box on the 5′ side Many RNA Pol II lack a TATA box or an Inr or both. additional sequences around TATA and downstream (to the right) of Inr may be recognized by one or more TFI
RNA polymerase II
in nucleoplasm synthesizes precursors for mRNA and small RNA molecules for the splicing apparatus has a long carboxyl terminal domain (CTD) that has many repeats of a "consensus heptad AA sequence" YSPTSPS (27 repeats in yeast, 52 in mice and humans) regulated by phosphorylation on Ser of CTD, this enhances transcription and recruits other factors required to process RNA Pol II product
How does this combination hairpin—oligo(U) terminate transcription?
First, RNA polymerase appears to pause immediately after it has synthesized a stretch of RNA that folds into a hairpin. Second, the RNA-DNA hybrid helix produced in the oligo(U) tail is unstable because rU-dA base pairs are the weakest of the three kinds of Watson—Crick base pairs. Hence, the pause in transcription caused by the hairpin permits the weakly bound nascent RNA to dissociate from the DNA template and then from the enzyme. The solitary DNA template strand rejoins its partner to re-form the DNA duplex, and the transcription bubble closes.
which direction does RNA transcribe?
RNA polymerase initiates and elongates the RNA product, with the chain growing in the 5' to 3' direction:
list features unique to transcription in eukaryote
1) More-Complex Transcriptional Machinery/Regulation RNA synthesis in eukaryotes is carried out by 3 distinct RNA polymerases, which recognize specific promoters there are many different types of promoter elements, which can combine in a multitude of ways, greatly increasing the number of types of promoters. access to eukaryotic promoters is restricted by the structure of chromatin, and activation of transcription is associated with many changes in chromatin structure in the transcribed region although eukaryotic cells have both positive and negative regulatory mechanisms, positive mechanisms predominate in all systems characterized so far (virtually every eukaryotic gene requires activation in order to be transcribed) eukaryotic cells have larger, more complex multimeric regulatory proteins than do bacteria. 2) RNA Processing: eukaryotes process nascent RNA very extensively into mature mRNA 3) The Nuclear Membrane: in eukaryotes, transcription takes place in the nucleus, whereas translation takes place outside the nucleus in the cytoplasm.
what are the 3 RNA polymerases?
1) RNA polymerase II (Pol II) in nucleolus (large structure in nucleus and serves as the site of ribosome synthesis and assembly. 2) RNA polymerase I (in nucleoplasm) 3) RNA polymerase III ( in nucleoplasm)
Successful binding of active RNA polymerase II holoenzyme at one of its promoters usually requires the action of other proteins, of four types:
1) Transcription activators, which bind to enhancers and facilitate transcription; (2) Chromatin modification and remodeling proteins (3) Coactivators (4) Basal transcription factors required at every Pol II promoter
RNA polymerase (AKA DNA-dependent RNA polymerase )
1)does not require a primer RNA sequence is complementary to DNA The DNA strand that has the same sequence as the RNA product (with T instead of U) is called the coding strand. 2) Activated precursors in the form of the 4 ribonucleoside triphosphates. 3) Divalent metal ions, usually Mg2+ or Mn2+.
RNA Polymerase mechanism
5'->3' The 3'OH of the growing RNA chain attacks the inner most phosphoryl (α) group of the incoming ribonucleoside triphosphate. This is essentially the same mechanism used by DNA polymerases
describe the role
A promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, and can be about 100-1000 base pairs long Promoters direct RNA polymerase to the proper initiation site for gene transcription.
transcription and translation in bacteria vs. eukaryotes
A) In bacteria, the primary transcript serves as mRNA and is used immediately as the template for protein synthesis. (B) In eukaryotes, mRNA precursors are processed and spliced in the nucleus before being transported to the cytoplasm for translation.
Dna and rna stop signals
DNA is a palindromic (inverted repeat) GC-rich region followed by a sequence of T residues. TTTTTT The RNA transcript of this DNA palindrome is self-complementary ⇒ forms a hairpin structure with a stem and loop •this stable hairpin is followed by a sequence of four or more uracil residues, which also are crucial for termination. The RNA transcript ends within or just after them UUUUUUU A termination signal found at the 3′ end of an mRNA transcript consists of a series of bases that form a stable stem-and-loop structure and a series of U residues.
and structure of promoters (the 2 conserved sequences)
In E. coli, 2 DNA sequences that are each 6 bp long act as a promoter for many genes the: -10 sequence and the -35 sequence (the consensus promoter sequences refer to the CODING STRAND OF DNA, the strand that mRNA looks like). 1) The -10 consensus sequence (TATAAT) used to be called the Pribnow box 2) -35 consensus sequence (TTGACA) was called the TATA box. Eukaryotes also have a TATAAA sequence, called a TATA box in the -25 region. The first nucleotide (the start site) of a transcribed DNA sequence (gene) is denoted as +1 and the second one as +2 (DOWNSTREAM); the nucleotide preceding the start site is denoted as −1 (UPSTREAM).
describe mechanisms for termination of transcription
In the termination phase of transcription, when RNA polymerase reaches a terminator sequence, RNA synthesis halts, the RNA-DNA hybrid dissociates, the melted region of DNA reanneals, and RNA polymerase releases the DNA. The free polymerase can, in principle, bind any σ subunit. The type bound determines the promoter to which the RNA polymerase will bind in the next round of synthesis
Binding and initiation phase
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 ~17 bp region of DNA is then unwound to form an open complex. the initiation phase encompasses transcription initiation and promoter clearance. once elongation commences, the σ subunit is released and the polymerase leaves the promoter and becomes committed to elongation of the RNA.
common eukaryotic promoter elements
Inr: initiator sequence (DNA is initially unwound) DPE: downstream promoter element Enhancer: separate from the promoter region, enhancer elements bind specific transcription factors. TATA box is the major assembly point for the proteins of the preinitiation complexes of Pol II:
Protein dependent termination
Protein-dependent termination requires the participation of an additional protein with ATPase activity called rho (ρ). hexameric ρ protein rho recognizes sequences located in the nascent RNA that are rich in cytosine and poor in guanine, and it specifically binds a stretch of 72 nucleotides on the nascent RNA in such a way that the RNA passes through the center of the structure. when ρ collides with the RNA polymerase at the transcription bubble, it breaks the RNA-DNA hybrid helix, unwinding the hybrid helix and stopping transcription.
mRNA, DNA Template, DNA Coding strand
The base sequence of mRNA (green) is the complement of that of the DNA template strand. The other strand of DNA is called the coding strand because it has the same sequence as that of the RNA transcript except for thymine (T) in place of uracil (U).
How is transcription regulated?
The efficiency, or strength, of a promoter sequence serves to regulate transcription: genes with strong promoters are transcribed frequently genes with very weak promoters are transcribed about once in 10 minutes
recognize the convention for numbering the nucleotides in the DNA template with regard to the transcription start site
The first nucleotide (the start site) of a transcribed DNA sequence (gene) is denoted as +1 and the second one as +2 (DOWNSTREAM); the nucleotide preceding the start site is denoted as −1 (UPSTREAM).
describe the model for the transcription bubble
The region containing the RNA polymerase, DNA, and the RNA product is called the transcription bubble.~17 bp of DNA are unwound at a time throughout the elongation phase, the same as in the initiation phase. Duplex DNA is unwound at the forward end of RNA polymerase and rewound at its rear end by RNA polymerase itself The newly synthesized RNA forms a hybrid helix with about 8 bp of the template DNA strand. The 3′-hydroxyl group of the RNA in this hybrid helix is positioned so that it can attack the α-phosphorus atom of an incoming ribonucleoside triphosphate. RNA polymerase continues transcription until a termination signal is reached. The elongation phase of RNA synthesis begins after the formation of the first phosphodiester linkage. an important change is the loss of the σ subunit shortly after initiation. The loss of σ enables the core enzyme to strongly bind to the DNA template
describe the structure and regulation of the lac operon in E. coli
The regulation of transcription from the lac operon illustrates how transcription in bacteria responds to environmental informatio See written notes
what is transcription and what is the enzyme that catalyzes it?
The synthesis of RNA from a DNA template a process catalyzed by RNA polymerase.
types of RNA
Three major types of RNA are produced in all cells: Messenger RNA (mRNA) encodes the information for the synthesis of a protein Transfer RNA (tRNA) and ribosomal RNA (rRNA) play key roles in translating mRNA information into protein (protein synthesis
cis-acting elements
are DNA sequences that regulate the expression of a gene located on the same molecule of DNA. A promoter and the gene that it regulates are always on the same molecule of DNA.
Trans-acting elements
are proteins that recognize cis-acting elements and regulate RNA synthesis. Transcription factors are trans-acting elements
list possible modifications of the transcription product into the mature RNA molecule in bacteria
mRNA no modifications after synthesis by RNA polymerase Transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules are generated by cleavage and other modifications of the transcription product 1) modification by splicing in E. coli, three kinds of rRNA molecules and a tRNA molecule are excised from a single primary RNA transcript that also contains noncoding regions called spacer regions Specific nucleases cleave and trim these precursors of rRNA and tRNA with high precision A) nucleases, b) ribonuclease 2 c) ribonuclease P 2) Modification by addition of nucleotides to the termini of some tRNA strands. For example, CCA, a terminal sequence required for the function of all tRNAs, is added to the 3′ ends of tRNA molecules that do not already possess this terminal sequence. 3) Modification by base alteration is a third way to alter rRNA and tRNA. Some bases of rRNA are methylated.
describe the role of the sigma (σ) subunit of RNA polymerase from E. coli.
sigma subunit helps find a site where transcription begins, participates in the initiation of RNA synthesis, and then dissociates from the rest of the enzyme. The enzyme responsible for transcription in all organisms is RNA polymerase. in E. coli, RNA polymerase holoenzyme is a very large (∼500 kd) and complex enzyme consisting of five kinds of subunits with the composition α2ββ′σω the sigma (σ) subunit helps to find a site where transcription begins, participates in the initiation of RNA synthesis, and then dissociates from the rest of the enzyme. RNA polymerase without this subunit (α2ββ′ω) is called the core enzyme. The core enzyme contains the active site. the σ subunit decreases the affinity of RNA polymerase for general regions of DNA by a factor of 104. This decrease has the effect of allowing the enzyme once bound to the DNA double helix to rapidly slide along it, searching for the promoter. Once the promoter is found and transcription begins, the σ subunit dissociates, allowing the core enzyme to bind tightly to DNA
RNA Pol II requires an array of proteins called
transcription factors, in order to form the active transcription complex. TFII are the general transcription factors required at every Pol II promoter (TFII) are highly conserved in all eukaryotes