11.RNA synthesis and processing

RNA合成和修饰RNA在细胞中起着极为重要的作用，它参与了基因表达的各个环节，包括转录、剪接、翻译等。

本文将探讨RNA的合成和修饰过程，在这些过程中起关键作用的主要因子和机制。

一、RNA的合成RNA的合成过程即转录过程，是将DNA模板上的基因信息转移到RNA分子上的过程。

转录是由RNA聚合酶（RNA polymerase）催化完成的。

1．转录起始转录的起始是指RNA聚合酶结合到DNA模板上的起始点，该点通常位于基因的启动子区域。

RNA聚合酶以DNA为模板合成RNA链，由酶促反应催化。

2．转录延伸转录延伸是指RNA聚合酶沿DNA模板逐渐进行合成RNA链的过程。

这个过程中，RNA聚合酶会读取DNA模板链的碱基序列，并将其转录成RNA链的互补序列。

该过程通常在核糖体RNA合成过程中发生。

3．转录终止转录终止是指RNA聚合酶在合成RNA链之后，从DNA模板上解离开来，结束合成过程。

在细菌中，转录终止通常由转录终止因子识别特定DNA序列引发，而在真核生物中，转录终止则与多个蛋白质因子相互作用。

二、RNA的修饰RNA合成后，还需要经过一系列的修饰过程，以使其成为功能成熟的RNA分子。

1．剪接修饰剪接修饰是指在真核生物的转录产物中，将内含子序列剪切除去，仅保留外显子序列的过程。

此过程主要由剪切体（spliceosome）完成，其中包含RNA分子和蛋白质组成的复合物。

2．3'端加工3'端加工是指RNA链的3'末端修饰，包括poly(A)尾部的加入。

poly(A)尾部是由多聚腺苷酸连接而成的结构，它有利于RNA分子的稳定性和转运。

3．修饰碱基的加入和修饰RNA分子中的某些碱基还可能被修饰，包括甲基化、核苷酸化等化学改变。

这些修饰可以影响RNA的稳定性、可折叠性和功能。

4．RNA修饰的功能RNA修饰可以影响RNA的翻译、稳定性和可折叠性，从而调节基因表达的水平。

此外，一些RNA修饰也可以作为细胞信号传递的重要分子。

RNA合成和RNA处理的分子机制RNA是生物中至关重要的分子之一，也是最为复杂、最为多样的一类分子。

RNA分子有多种类型，从结构上可以分为单链RNA和双链RNA两大类；从功能上可以分为编码RNA（mRNA）、转运RNA（tRNA）、核糖体RNA（rRNA）和调控RNA等多个亚类别。

而RNA分子的活动都建立在RNA合成和RNA处理的基础之上，本文将介绍这两个分子机制的基本过程和分子机理。

一、RNA合成RNA合成是指在核糖体DNA模板上，RNA聚合酶（RNA polymerase）酶催化下，将分子游离核苷酸按照序列顺序逐个加入RNA链的过程。

该过程包括三个步骤：初始化、延伸和终止。

初始化是RNA聚合酶找到DNA链的起始点，与DNA 发生比较弱的互作用，形成一个封闭结构（closed complex）。

随着RNA聚合酶取消封闭，DNA的双链被分离，核苷酸三磷酸单元以骨架糖-磷酸形式逐个加入RNA链，完成RNA的延伸，直至终止。

RNA合成的机理可以分为两个过程：核苷酸互补匹配和酯合反应。

核苷酸互补匹配是指DNA和RNA在一定条件下，由于碱基间的互补配对而能稳定结合。

RNA合成过程中，RNA分子的含氧组件（-OH）与核苷三磷酸分子的α磷酸（-PΟ4）发生酯合反应，同时释放出氢氧离子（H+）和二氧化碳（CO2），形成一条新的RNA链。

在细胞中，mRNA的合成过程常常要经过多种类型的调控，以保持细胞内同步和相互协调的转录事件。

这些调控机制分为两大类型：转录因子和表观遗传机制。

转录因子是指一类能够与RNA聚合酶直接或间接结合，并调控转录起始和终止的蛋白质。

表观遗传机制则指通过DNA和蛋白质修饰等方式，调控DNA表达和RNA合成的复杂生物化学过程。

二、RNA处理RNA处理指的是RNA聚合酶合成的RNA分子，在核糖体DNA模板上被合成出来后，需要进一步经过核内和核外的修饰和加工，才能成为细胞中不同类型RNA的功能分子。

Chapter five RNA synthesis and RNA synthesis and processing(RNA合成与加工）(ARNA synthesis isll dOverviewcalled transcription (转)录).Transcription is aprocess in whichprocess in which one DNA strand isused as template to psynthesize acomplementaryRNA.I t li ll l1%f th DNA iIn most mammalian cells, only 1% of the DNA sequence is copied into a functional RNA (mRNA). Only one part of the DNA is transcribed to produce nuclear RNA, and only a minor portion is transcribed to produce nuclear RNA and only a minor portion of the nuclear RNA survives the RNA processing steps.One of the most important stages in RNA processing is RNA splicing (RNA剪接). In many genes, the DNA sequence coding splicing(RNA In many genes,the DNA sequence coding for proteins, or "exons", may be interrupted by "introns".In the cell nucleus, the DNA that includes all the exons andIn the cell nucleus the DNA that includes all the exons and introns of the gene is first transcribed into a complementary RNA copy called "nuclear RNA," or nRNA. In a second step, introns py,p,are removed from nRNA by RNA splicing. The edited sequence is called "messenger RNA," or mRNA.T l t t d d di t dTemplate strand and coding strandTo distinguish the two strands of DNA –p•The template strand refers to the strand of DNA that serves as a template for the synthesis of mRNA.The coding strand (编码链)or the mRNA like strand •The coding strand(or the mRNA-like strand refers to the strand with a base sequence directly corresponding to the mRNA sequence.“Template strand (模板链) ”may be called “minus strand”, or “antisense strand (反意义链) ”.The "coding strand"may be termed "non-template strand", p,"plus strand", or "sense strand".RNA structure1.Covalent structure: like DNA, RNA is a long polymer consisting of nucleotides joined by 3, 5phosphodiester consisting of nucleotides joined by3’5’phosphodiester bonds. However, there are some differences:•The bases in RNA are Adenine, Guanine, Uracil andTh b i RNA Ad i G i U il d Cytosine. Thymine in DNA is replaced by Uracil in RNA. The uracil can form base pairs with adenineThe uracil can form base pairs with adenine.•Sugar in RNA is ribose rather than deoxyribose as in DNA2.As with DNA, the nucleotide sequence of RNA is also written as a base sequence in the 5’→3’direction. Thusw tte as a base seque ce t e53d ect GUCAAGCCGGAC is the sequence of one short RNA molecule.3. RNA secondary structureyMost RNA molecules are single-stranded but an RNA molecule may contain regions which can form complementary base paring may contain regions which can form complementary base-paring where the RNA strand loops back on itself.Classes of RNA （RNA的种类）Classes of RNA1) Messenger RNAs (mRNAs, 信使RNA):This class of RNAs is the genetic coding templates used by This class of RNAs is the genetic coding templates used by the translational machinery.2) Transfer RNAs (tRNAs, 转运RNA):This class of small RNAs forms covalent attachments to individual amino acids and recognizes the encoded sequences of the mRNAs.f h3)Ribosomal RNAs (rRNAs, 核糖体RNA):This class of RNAs is assembled, together with numerous ribosomal proteins, to form the ribosomes (核糖体).4) Small nuclear RNAs (snRNA,小核RNA):4)Small nuclear RNAs(snRNA RNA):This class of RNA is part of the machinery for splicing precursor mRNA. It is only present in eukaryote. precursor mRNA It is only present in eukaryoteUnit one: RNA Polymerases(RNAy(聚合酶) 1.The function of RNA polymerases• A single RNA polymerase catalyzes the synthesis of all threeE. coli RNA classes--mRNA, rRNA, and tRNA.•RNA polymerases can initiate a new strand.•The nucleotides used to extend a growing RNA chain Th l id d d i RNA h i are ribonucleoside triphosphates(NTPs).①Two phosphate groups are released as pyrophosphate(PP)during the reaction.焦磷酸) during the reaction. pyrophosphate (PPi②Strand growth is always in the 5' to 3'direction.③The first nucleotide at the 5' end retains its triphosphate group.p p g pThe chemical reaction catalyzed by RNA polymerases. The chemical reaction catalyzed by RNA polymerases2. Transcription exhibits several features thatare distinct from replication.）Transcription initiates, both in prokaryotes and1Transcription initiates both in prokaryotes and eukaryotes, from many more sites than replication.2）There are many more molecules of RNA polymerase per cell than DNA polymerase.3）RNA polymerase proceeds at a rate much slower than DNA polymerase (approximately 50-100 bases/sec for RNA DNA polymerase(approximately50-100bases/sec for RNAversus near 1000 bases/sec for DNA).4）The fidelity of RNA polymerization is much lower than DNA.3Classes of RNA polymerases3. Classes of RNA polymerases)y y1)Prokaryotic RNA Polymerase（E.coli）:The σsubunit (σfactor) plays an important role in recognizingthe transcriptional initiation site, and also possesses the helicaseth t i ti l i iti ti it d l th h li activity to unwind the DNA double helix.Several different forms of σsubunits have been identified, with molecular weights ranging,g g g from 28 kDa to 70 kDa.y y p yNucleotide synthesis is carried out by core polymerase.The "holoenzyme"refers to a complete and fully functionale y e.e o oe y e c udes e co e po y e se d enzyme.The holoenzyme includes the core polymerase andthe σfactor.Once the "holoenzyme"recognizes the correctrecognizes the correctpromoter, the sigma factord ssoc es oe Ndissociates from the RNApolymerase.The core polymeraseThe core polymerasebegins to unwind the helixof the DNA creating aregion of non-paireddeoxyribonucleotides thatserve as a template fort l t fRNA synthesis.2)EukaryotesIn eukaryotes, the genetic information carried by DNA is containedwithin the nucleus. This information is transcribed into RNA which mustthen be carried across the membrane into the cytosol prior to translation. then be carried across the membrane into the cytosol prior to translation This spatial and temporal separation of transcription and translation isone of many differences between eukaryotic and prokaryoticone of many differences between eukaryotic and prokaryotictranscription.Eukaryotic cells have three kinds of RNA1RNA Polymerase I (Pol I)Polymerases1.RNA Polymerase I (Pol I)rRNAPromoters vary greatly2RNA Polymerase II (Pol II)2. RNA Polymerase II (Pol II)mRNAPromotor -TATA box (TATAAA) and Inr Initiator3. RNA Polymerase III (Pol III)y ()tRNA, 5S rRNAα鹅膏蕈碱g yUnit two: Gene's Regulatory ElementsTranscriptional regulation is mediated by the interaction between transcription factors and their DNA binding sites.Trans-acting element (反式作用元件):sequences encoding •Trans acting element(sequences encoding transcription factors•Cis-acting elements (顺式作用元件): DNA sequences that are binding sites of transcription factorsThe cis-acting elements may be divided into four types:1. Promoter (启动子)The promoter region contains important sequences that are required for RNA polymerase to bind. These sequences are q p y qsimilar in both prokaryotic and eukaryotic genes, but the locations are different.C ll T L ti S Conserved Sequences in Promoters:Cell Type LocationSequence -10TATAAT Prokaryotic10-35TTGACA Eukaryotic about -25about -80TATA (TATA box)CAAT (CAAT box))1)Promoter site in E.coli•The first nucleotide of transcribed sequence is always referred to as ＋1.as1•The two promoter elements are located at positions –10 and –35 and called the –10 sequence and –35 sequence.d ll d h10d352) Promoter elements(启动子元件)in eukaryotic protein genest i:•TATA box is located at -35 to -20.•Promoter-proximal elements (启动子近侧元件), such as CAAT box and GC box,are often located within 200 bp of the transcriptional start site.Promoter-proximal elements()p•initiator (Inr, 起始子): another promoter elementInr has the consensus sequence:PyPy A N(T/A)PyPy.I h th P P N(T/A)P PPy denotes pyrimidine (C or T), N = any, (T/A) means T or A. The base A at the third position is located at +1 (the transcriptional start site).Core promoter elements: TATA box and initiatorTBP:TATA-box binding protein is essential for the initiation of transcription. It forms a central part of the "pre-initiation complex".2. Enhancers (增强子)Enhancers are the positive regulatory elements, which are located either upstream or downstream of the transcriptional located either upstream or downstream of the transcriptional initiation site, or even within the gene they control. of E coli glnA In prokaryotes enhancers are quite close to the promoter.•Enhancers of E. coli glnA gene•Enhancers of eukaryotesEukaryotic enhancers could be far from the promoter. Enhancer-binding proteins, in addition to their DNA-binding site, Enhancer binding proteins in addition to their DNA binding sitehave sites that bind to transcription factors ("TF") assembled atp gthe promoter of the gene.3. Silencers(静息子／沉默子)Silencers are the DNA elements which interact with repressors (阻遏物) to inhibit transcription.Silencers may be located thousands of base pairs away from the gene they control.In a few cases, a DNA element may act either as enhancer or silencer, depending on the binding protein.silencer depending on the binding protein4. Response elementsResponse elements are the recognition sites of certain transcription factors.Most of them are located within 1 kb (kilo base-pair) from the transcriptional start site. Eukaryotic response elements:Eukaryotic response elements:Response Element Transcription Factor Consensus SequenceTGACGTCACRE CREB(cAMP-response-element-binding protein)g pERE Estrogen receptor AGGTCANNNTGACCTGRE Glucocorticoid receptor AGAACANNNTGTTCTHSE Heat shock factor GAANNTTCNNGAASRE Serum response factor CC(A/T)GG6Unit three:Transcription ProcessUnit three: Transcription Process Transcription and Translation (翻译) in Cells •Transcription and Translation()in CellsIn a prokaryotic cell, transcription and translation are In a prokaryotic cell transcription and translation are coupled; that is, translation begins while the mRNA is still being synthesized.still being synthesizedIn a eukaryotic cell, transcription occurs in theIn a eukaryotic cell transcription occurs in the nucleus, and translation occurs in the cytoplasm.Prokaryotic Celle e s o uc eus o sep e e p ocesses o sc p o d There is no nucleus to separate the processes of transcription and translation.When bacterial genes are transcribed their transcripts can When bacterial genes are transcribed, their transcripts can immediately be translated.Eukaryotic Cells•Transcription and translation are spatially and temporally separated; that is, transcription occurs in the nucleus to produce a pre-mRNA molecule. •The pre-mRNA is processed to produce the mature mRNA, which exits the •The pre mRNA is processed to produce the mature mRNA which exits the nucleus and is translated in the cytoplasm.•Transcription in prokaryotesThree phases of transcription1. Initiation(起始)1I iti ti(i)Binding of polymerases to the initiation site.Promoter sequences promote the ability of RNA polymerases to recognize the nucleotide at which initiation begins.to recognize the nucleotide at which initiation begins. Enhancers enhance polymerase activity even further.In both prokaryotic and eukaryotic transcription,the first p p p incorporated ribonucleotide is a purine and it is incorporated as a triphosphate.Every gene has a promoter, which is the binding site for the basal transcriptional apparatus -RNA polymerase and its co-factors. This provides the minimum machinery necessary to allow transcription of the gene.(ii)Unwinding (melting) of the DNA double helixIn prokaryotes, binding of the polymerase s σfactor toIn prokaryotes,binding of the polymerase's factor to promoter can catalyze unwinding of the DNA double helix.)block RNA chain initiation Rifamycins(利福霉素) block RNA chain initiation.2.Elongation(延长)2El ipAfter the DNA strands have been separated at thepromoter region, the core polymerase can start tosynthesize RNA based on the sequence of the DNAtemplate strand.l dActinomycin D(放线菌素D)inhibits elongation by intercalating (插入) into the template DNA helix.3Termination(3. Termination (终止)Following termination,the core polymerase dissociates from the template.the templateThe core and sigma subunit can then reassociate forming the holoenzyme again ready to initiate another round ofh l i d i i i h d f transcription.In prokaryotes, the transcription is terminated by two major mechanisms:h i①Rho-independent termination.Two structural features of all E coli rho independently terminating Two structural features of all E. coli rho-independently terminating genes have been identified.One feature is the presence of symmetrical GC-rich segments that O f t i th f t i l GC i h t th t are capable of forming a stem-loop (茎环) structure in the RNA. The second is a downstream A rich sequence in the template.h d i d i h i h lp q p②Rho-dependent termination requires the termination protein, rho.The rho factor recognizes and binds to termination sequences in the 3' portion The rho factor recognizes and binds to termination sequences in the3'portion of the RNA.This binding destabilizes the polymerase-template interaction leading toThis binding destabilizes the polymerase template interaction leading todissociation of the polymerase and termination of transcription.http://vcell ndsu edu/animations/transcription/movie-/animations/transcription/movie-flash.htmRNA processingp gIn prokaryotes, mRNA transcribed from protein-coding genes requires little or no modification prior to translation. tRNA and requires little or no modification prior to translation tRNA and rRNA are synthesized as precursor.The precursor is cleaved by specific ribonucleases to yield mature 23S, 16S and 5S rRNA as well as single tRNA.d ll i lThe three nucleotides CCA are added to the 3’end of tRNA before they can function.。

RNA的生物合成与加工贮存于DNA中的遗传信息通过转录和翻译而得到表达。

在转录过程中，DNA的一条链作为模板，在其上合成出RNA分子，合成以碱基配对的方式进行，所产生的RNA链与DNA模板链互补。

细胞各类RNA，包括合成蛋白质的mRNA、ｒRNA、ｔRNA，以及具有各种特殊功能的小RNA，都以DNA为模板，在RNA聚合酶催化下合成的，最初转录的RNA也要经过一系列加工和修饰才能成为成熟的RNA分子。

RNA所携带的遗传信息也可以用于指导RNA或DNA的合成，前一过程即RNA复制，后一过程为逆转录。

由于RNA既能携带遗传信息，又具有催化功能，故推测生命起源早期存在RNA世界。

DNA指导下的RNA合成在DNA指导下RNA合成称为转录，RNA链的转录起始于DNA模板的一个特定起点，并在另一中指点终止。

此转录区域为转录单位。

转录单位可以是一个基因，也可是多个基因。

基因是遗传物质的最小功能单位，相当于DNA的一个片段。

它通过转录对表型有专一性的效应，并可突变成各种形式。

极影的转录是一种有选择性的过程，随着细胞的不同生长发育阶段和细胞内外环境的改变而转录不同的基因。

转录的起点是由DNA的启动子来控制的，控制终止的部位则称终止子。

转录是通过DNA知道的RNA聚合酶来实现。

DNA指导的RNA聚合酶该酶需要以四种核糖核苷酸作为底物，并需要适当的DNA作为模板，镁离子能促进聚合反应。

RNA链的合成方向也是5’-3’，第一个核苷酸带有三三个磷酸基，其后每加入一个核苷酸脱去一个焦磷酸，形成磷酸二酯键，反应是可逆的，但焦磷酸的分解可推动反应趋向聚合。

与DNA聚合酶不同，RNA聚合酶无需引物，它能直接在DNA模板上合成RNA链，但是RNA 聚合酶无校对功能。

分子杂交实验表明，合成的RNA只与模板DNA形成杂交体，而与其他DNA不能形成杂交体，这就说明反应产物RNA是在作为模板的DNA上，通过碱基配对机制合成的。

在体外，RNA聚合酶能使DNA 的两条链同时进行转录，但在体内的形况不同，许多实验证明，在体内DNA两条链中仅有一条链可用于转录，或某些区域以这条链转录，另一些区域以另一条链转录，用于转录的链叫做模板链，或负链。