Molecular Biology Chen Weiwen Department of Biochemistry and Molecular Biology，school of basic medical sciences,Shandong University All pictures of this PPT are from the network Chapter 14 Biosynthesis of RNA 1．About transcription，which of the following is TRUE? A．use DNA as template B．use RNA as template C．catalyzed by DNA pol D．use dNTPs as precursor E．take place in cytoplasm 2. The initiation of transcription requires an RNA polymerase binding to DNA at_____ A．CAAT box B．enhancer C．promoter D．HRE E．silencer RNA Synthesis Transcription: DNA –dependent RNA Synthesis A T G C A U G C RNA replication: RNA –dependent RNA Synthesis (RNA virus) Section I Templates & Enzymes in prokaryotic transcription ① Materials involving in transcription: Substrate: NTPs (ATP, GTP, CTP, UTP) Template: DNA Enzyme: DNA dependent RNA polymerase (RNA pol) Others: protein factors, Mg2+ , Zn2+ ②RNA synthesis in the 5’→3’ direction ③NMPs are linked by 3’,5’ – phosphodiester bonds to form RNA molecule I Template for transcription in prokaryotes 1. Only one of the two DNA strands serves as a template. 2. The strand that serves as template for RNA synthesis is called the template strand. 3. The DNA strand complementary to the template strand is called the nontemplate strand/ coding strand. By convention, DNA sequences are shown as they exist in the nontemplate strand, with the 5’ terminus on the left. II RNA Is Synthesized by RNA Polymerases 1. Action mode of RNA polymerases ①Chemical mechanism of RNA synthesis ②chemical reaction formula ③Each nucleotide in the newly formed RNA is selected by Watson-Crick base-pairing interactions ④Does not require a primer to initiate synthesis. ⑤RNA pol requires DNA for activity and is most active when bound to a ds DNA. ⑥In combination with a specific sequence of DNA, promoters, RNA synthesis can be initiated，but only one of the two DNA strands serves as a template. 2. Structure of the RNA polymerase (E. coli) a large, complex enzyme core enzyme: α2ββ’ω Core enzyme has the ability to synthesize RNA on a DNA template, but it cannot recognize promoters. holoenzyme: α2ββ’ ωσ σ binds transiently to the core and directs the enzyme to specific binding sites on the DNA The holoenzyme exists in several forms, depending on the type of σ Structure of the RNA polymerase core enzyme of E. coli The most common subunit is σ70 the promoters of the heat shock genes are recognized and bound with by σ32 Rifampicin inhibits bacterial RNA synthesis by binding to the β subunit, preventing the promoter clearance. Ⅲ RNA Synthesis Begins at Promoters Asymmetric transcription RNA is transcribed from only one of the DNA strands that serves as the template The template strand of different genes is not always on the same strand of DNA Only part of the DNA sequence is transcribed The importance of RNA polymerase orientation The direction of transcription is determined by the promoter at the beginning of each gene (green arrowheads). Prokaryotic transcription units are called operons A cluster of bacterial genes can be transcribed from a single promoter. RNA pol binds to promoters, which direct the transcription of adjacent segments of DNA (genes) How to determine the sequence of the promoter? RNA Polymerase Leaves Its Footprint on a Promoter Footprinting: a technique derived from principles used in DNA sequencing, identifies the DNA sequences bound by a particular protein. RNA polymerase protection method Footprint analysis of the RNA polymerase –binding site on a DNA fragment Footprinting results of RNA polymerase binding to the lac promoter By convention, DNA sequences are shown as they exist in the nontemplate strand, with the 5’ terminus on the left. Nucleotides are numbered from the transcription start site (TSS), with positive numbers to the right (in the direction of transcription) and negative numbers to the left. Prokaryotic promoters contain consensus sequences Analyses and comparisons of the most common class of bacterial promoters (σ70) have revealed similarities in two short sequences centered about positions -10 and -35 consensus sequence are important interaction sites for the σ70 subunit Consensus nucleotide sequence for the major class of E. coli promoters A sequence logo displaying the information of consensus sequence The consensus sequence at the -10 region is (5’)TATAAT(3’). The consensus sequence at the -35 region is (5’)TTGACA(3’). A third AT-rich recognition element, called the UP (upstream promoter) element, occurs between positions -40 and -60 in the promoters of certain highly expressed genes. The UP element is bound by the α subunit of RNA polymerase. Typical E. coli promoters recognized by an RNA polymerase holoenzyme containing σ70 Pribnow box The promoter sequence determine a basal level of expression that can vary greatly from one E. coli gene to the next. The σ subunit is a specificity factor that mediates promoter recognition and binding. When bound to σ32, RNA polymerase is directed to a specialized set of promoters with a different consensus sequence Section II. The Process of Transcription in Prokaryotes Initiation Elongation Termination I. Initiation Step 1: A closed transcription complex is formed. Step 2: An open transcription complex forms in succession. Step3: the formation of the first 3’,5’- phosphodiester bond. Step 1: A closed transcription complex is formed. Step 2: an open transcription complex form in succession. A ~17 bp region from within the -10 to +2/+3. Step3: the formation of the first 3’,5’- phosphodiester bond. Mostly GTP/ATP Points of transcription initiation Holoenzyme required Primer not required First nucleotide often is GTP/ATP: 5’- pppGpN-OH3’ II. Core enzyme elongate the RNA chain independently 1. promoter clearance ① Abortive initiation is overcome ② The σ subunit dissociates as the polymerase enters the elongation phase. ③ transcription complex move away from the promoter. 2. Transcription bubbble DNA duplex unwind about 17 bp distance by core enzyme movement, forming a transcription “bubble.” the growing 3’ end of the new RNA strand base- pairs temporarily with the DNA template to form a short hybrid RNA-DNA double helix (8 bp) G≡C > A=T > A=U The RNA in this hybrid duplex “peels off” shortly after its formation, and the DNA duplex re-forms. 50 nt/s Two-dimensional image of an elongating bacterial RNA polymerase, as determined by atomic force microscopy Summerize for elongation Core enzyme required RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3’-hydroxyl end, building RNA in the 5’→3’ direction. The template DNA strand is copied in the 3 ’→ 5’ direction (antiparallel to the new RNA strand) Dynamic 8 bp RNA-DNA hybrid occurs in this unwound region (transcription bubble) The DNA is unwound ahead and rewound behind as RNA is transcribed. Ⅲ In bacteria, transcription and translation are tightly coupled 1. one gene usually is transcribed by many molecules of RNA polymerase simultaneously. 2. Each mRNA is being translated by many ribosomes simultaneously. Coupling of transcription and translation in bacteria Ⅳ E. coli has at least two classes of termination signals: one class relies on a protein factor ρ and the other is ρ- independent. 1.ρ-dependent termination ① The ρ -dependent terminators usually include a CA-rich sequence called a rut (rho utilization) element. ② The ρ protein has an ATP-dependent RNA- DNA helicase activity ③ The ρ protein associates with the RNA at rut site and migrates in the 5’→3’ direction until it reaches the transcription complex that is paused at a termination site ④ There it contributes to release of the RNA transcript ⑤ ATP is hydrolyzed by ρ protein during the termination process. ρ-dependent termination 2.ρ-independent termination ① Most ρ-independent terminators have two distinguishing features. First: a region that produces an RNA transcript with self-complementary sequences, permitting the formation of a hairpin structure centered 15 to 20 nucleotides before the projected end of the RNA strand. Second: a highly conserved string of A residues in the template strand that are transcribed into U residues near the 3’ end of the hairpin. ② When a polymerase arrives at a termination site with this structure, it pauses ③ Formation of the hairpin structure in the RNA disrupts several A=U base pairs in the RNA-DNA hybrid segment and may disrupt important interactions between RNA and the RNA polymerase, facilitating dissociation of the transcript. Palindromic DNA (or RNA) sequences can form alternative structures with intrastrand base pairing. termination signal sequence contains an inverted repeat followed by a stretch of AT base pairs (top). The inverted repeat, when transcribed into RNA, can generate the secondary structure in the RNA transcript (bottom). Formation of this RNA hairpin causes RNA polymerase to pause. ρ-independent termination The transcription cycle of bacterial RNA polymerase Section III The Biosynthesis of Eukaryote RNA I. Eukaryotic Cells At Least Have Three Kinds of Nuclear RNA Polymerases 1. Pol II transcribes most genes, including all those that encode proteins. 2. Pol II has many structural similarities to bacterial RNA pol Prokaryote Prokaryote & Eukaryote 3. Pol II (yeast) is a huge enzyme with 12 subunits (RPB1-12) 4. The largest subunit RPB1 has an carboxyl- terminal domain (CTD) ① CTD: a long tail consisting of many repeats of a consensus heptad amino acid Tyr-Ser-Pro-Thr- Ser-Pro-Ser ② The CTD has many important roles in Pol II function through its reversible phosphorylation ③ Phosphorylation of CTD allows multi-sets of proteins to associate with it that function in transcription elongation and RNA processing. Reversible phosphorylation of CTD CTD functions like the flight deck of aircraft carrier 5. Pol Ⅱ bind to Type Ⅱ 6. There are several important differences in the way in which the bacterial and eukaryotic RNA pol function Eukaryotic transcription initiation must take place on DNA that is packaged into nucleosomes and higher- order forms of chromatin structure , features that are absent from bacterial chromosomes.