Section M Transcription in Eukaryotes
一．Three eukaryotic polymerases
TypeRNA Pol IRNA Pol IIRNA Pol III
LocationNucleoli核仁SubstrateMost rRNAs gene (28, 18，5.8 S)?-amanitinInsensitiveNucleoplasm All protein-coding genes and some 核质snRNA genesNucleoplasm tRNAs, 5S rRNA,U6 snRNA and 核质other small RNAsVery sensitiveModerately sensitive二．RNA polymerase subunits
1.Each eukaryotic polymerase contains 12 or more subunits. 2. the two largest subunits are similar to each other and to the b’and b subunits of E.coli RNA Pol. 3.There is one other subunitα in all three RNA Pol homologous to a subunit of E. coli RNA Pol. 4. at least Five additional subunits are common to all three polymerases. 5.Each RNA Pol contains additional four or seven specific subunits.
三．The CTD of RNA pol II
1.The C-terminus of RNA Pol II contains a stretch of seven amino acids that is repeated 52 times in mouse enzyme and 26 times in yeast.
2.The heptapeptide sequence is: Tyr-Ser-Pro-Thr-Ser-Pro-Ser. These repeats are essential for viability.
3.This repeated sequence is known as carboxyl terminal domain (CTD)
4.The CTD sequence may be phosphorylated at the serines and some tyrosines
The CTD is unphosphorylated at transcription initiation, and phosphorylation occurs during transcription elongation as the RNA Pol II leaves the promoter (In vitro results).
6. Because it transcribes all eukaryotic protein-coding gene, RNA Pol II is the most important RNA polymerase for the study of differential gene expression. The CTD is an important target for differential activation of transcription elongation.
四．Ribosomal RNA Genes & nucleolus
1.A copy of 18S, 5.8S and 28S rRNA genes is organized as a single transcription unit in eukaryotes. A 45S rRNA transcript (~13 000 nt long) is produced during transcription, which is then processed into 18S, 5.8S and 28S rRNA.
2.Pre-rRNA transcription units are arranged in clusters in the genome as long tandem arrays separated by nontranscribed spacer squences.
3.Continuous transcription of multiple copies of rRNA genes by RNA Pol I is essential to produce sufficient rRNAs which are packaged into ribosomes.
4.The arrays of rRNA genes (rRNA cluster) loop together to form the nucleolus and are known as nucleolar organizer regions.
5.During active rRNA synthesis, the pre-rRNA transcripts are packaged along the rRNA genes, visualizing in the electronic microscope as “Christmas tree structures”.
五．RNA Pol I promoters
1.Mammalian pre-rRNA gene promoters have a bipartite sequence in the region preceding the start site, including core element and the upstream control elements (UCE). 2. RNA Pol I promoters in human cells are best characterized. ? Core element: -31 to +6, essential for transcription initiation.
? UCE: about 50-80 bp begins around 100 bp upstream from the start site, to increase the
transcription efficiency.
? Both regions are rich in G:C, with ~85% identity. 3.Upstream binding factor (UBF)
? A specific DNA-binding protein that binds to UCE, as well as a different site in the upstream
of the core element, causing the DNA to loop between the two sites. (two binding sites have no obvious similarity)
? UBF is essential for high level of transcription, and low level of expression occurs in its
absence.
六．RNA Pol III
? Contains at least 16 or more subunits ? Is located in nucleoplasm
? Synthesizes the precursors of 5S rRNA, the tRNAs and other small nuclear and cytosolic
RNAs 1.tRNA genes
? The initial transcripts of tRNA genes need to be processed to produce the mature tRNA.
? The transcription control regions of tRNA lies after the start site within the transcribed region.
The two highly conserved control sequences are called A box (5’-TGGCNNAGTGG) and B box (5’-GGTTCGANNCC). 2.Alternative RNA Pol III promoters
Many RNA Pol III genes also rely on upstream sequences for regulation of their transcription ? Use only regulatory genes upstream from their transcription start sites. 3.RNA Pol III termination
The RNA polymerase can terminate transcription without accessory factors. A cluster of A residue is often sufficient for termination.
七．RNA Pol II
? located in nucleoplasm
? catalyzing the synthesis of the mRNA precursors for all protein-coding genes.
? RNA Pol Ⅱ-transcribed pre-mRNAs are processed through cap addition, poly(A) tail
addition and splicing. 1.Promoters
① Most promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription. It has a 7 bp consensus sequence 5’-TATA(A/T)A(A/T)-3’.
② TBP binds to TATA box that includes an additional downstream bp.
③ TATA box acts in a similar way to an E. coli promoter –10 sequence to position the RNA Pol II for correct transcription initiation. The spacing but not the sequence between the TATA box and the start site is important. Transcription starts with an adenine ~50% of the time.
④ Some eukaryotic genes contain an initiator element instead of a TATA box. The initiator element is located around the transcription start site.
⑤ Other genes have neither a TATA box nor an initiator element, and usually are transcribed at very low rates.
2.Upstream regulatory elements
? The basal elements (the TATA box and initiator elements) : primarily determine the location
of the startpoint, and sponsor initiation only at a rather low level.
? Upstream regulatory elements (URE) such as the SP1 box and CCAAT boxes, greatly
increase the frequency of initiation. URE is located within 100-200 bp from the promoter, and plays an important role in ensuring efficient transcription. 3.Enhancers
1) Sequence elements which can activate transcription from thousands of base pairs upstream or
downstream.
2) General characteristics of Enhancers
? Exert strong activation of transcription of a linked gene from the correct start site. ? activate transcription when placed in either orientation with respect to linked genes
? Able to function over long distances of more than 1 kb whether from an upstream or
downstream position relative to the start site.
? Exert preferential stimulation of the closet of two tandem promoters
Section N
Regulation of transcription in eukaryotes
一．Eukaryotic Transcription Factors
1.Transcription factor domain structure ? DNA-binding domains
? Dimerization domains
? Transcription activation domains ? Repressor domains
2.Targets for transcriptional regulation
3. The activity of a transcription factor can be assigned to separate protein domains ? activation domains. (activity) ? DNA-binding domains. (activity)
? dimerization domains. Many transcription factors occur as homo- or heterodimers, held
together by dimerization domains. (regulation)
? ligand-binding domains. Allowing regulation of transcription factor activity by binding of an
accessory small molecule.The steroid hormone receptors are an example containing all for of these types of domain. (regulation)
4.Homeodomain proteins: The homeodomain (同源结构域) is a class of helix-turn-helix DNA-binding domain and recognizes DNA in essentially the same way as those bacterial proteins.
5.The zinc finger domain 1) C2H2 zinc finger 2) C4 zinc finger
6.Dimerization domains
① Leucine zippers
? Leucine zipper proteins contain a hydrophobic leucine residue at every seventh position in a
region that is often at the C-terminal part of the DNA-binding domain
? These leucines are responsible for dimerization through interaction between the hydrophobic
faces of the α-helices. This interaction forms a coiled-coil structure ② The helix-loop-helix domain (HLH)
? The overall structure is similar to the leucine zipper, except that a nonhelical loop of
polypeptide chain separates two α-helices in each monomeric protein.
? Hydrophobic residues on one side of the C-terminal α-helix allow dimerization. ? Example: MyoD family of proteins. 7.Repressor domains
1) Repression of transcription may occur by indirect interference with the function of an
activator. This may occur by:
2) Blocking the activator DNA-binding site (as with prokaryotic repressors)
Section O
RNA PROCESSING AND RNPs
一．RNA Processing
1.primary transcript 2.Romoval of nucleotides
Removal of nucleotides by both endonucleases and exonucleases
? endonucleases to cut at specific sites within a precursor RNA ? exonucleases to trim the ends of a precursor RNA
? This general process is seen in prokaryotes and eukaryotes for all types of RNA 3.addition of nucleotides to the 5’- or 3’- ends
Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products. ? Add a cap and a poly(A) tail to pre-mRNA 4.modification of certain nucleotides
Modification of certain nucleotides on either the base or the sugar moiety. ? Add a methyl group to 2’-OH of ribose in mRNA (A) and rRNA ? Extensive changes of bases in tRNA 5.mature RNA
二．RNPs
Ribonucleoproteins (RNP核糖核蛋白) = RNA protein complexes ? The RNA molecules in cells usually exist complexed with proteins ? specific proteins attach to specific RNAs
? Ribosomes are the largest and most complex RNPs
三．rRNA processing in prokaryotes
1.There are 7 different operons for rRNA that are dispersed throughout the genome.
2.Each operon contains one copy of each of the 5S,the 16S and the 23S rRNA sequences. About 1~4 coding sequences for tRNA molecules are also present in these rRNA operons.
RNase III, involved in the first step of rRNA processing
RNase M5, M16 and M23 are involved in the second step of rRNA processing
Step 1: Following or during the primary transcription, the RNA folds up into a number of stem-loop structures by base pairing between complementary sequences
Step 2: The formation of this secondary structure of stems and loops allows some proteins to bind to form a RNP complex which remain attached to the RNA and become part of the ribosome Step 3: After the binding of proteins, nucleotide modifications take place.
Example: methylation of adenine by methylating agent
Step 4: RNA cleavage
四．rRNA processing in eukaryotes
1.rRNA features
? The rRNA genes are present in a tandemly repeated cluster containing 100 or more copies of
the transcription unit, and are transcribed in nucleolus by RNA Pol I
? Precursor sizes are different among organisms (yeast: 7000 nt; mammalian 13500 nt), and
pre-mRNA processing is also slightly different among organism. ? The precursor contains
① one copy of the 18S coding region and
② one copy each of the 5.8S and 28S coding regions, which together are the equivalent of the 23S rRNA in prokaryote
? The large precursor RNA undergoes a number of cleavages to yield mature RNA and
ribosome.
? The eukaryotic 5S rRNA
① is transcribed by RNA Pol III from unlinked genes to give a 121nt transcript ② the transcript undergoes little or no processing
? Introns in rRNA genes of some lower eukarytes (Tetrahymena thermophila) must be spliced
out to generate mature rRNAs.
? Many introns are found to catalyze the splicing reaction by itself in vitro, therefore called
ribozyme
五.Ribosomes
1.Prokaryotic Ribosome 2.Eukaryotic Ribosome
3.The rRNAs and Ribosomes rRNAs in Prokaryotes: – 5S – 16S – 23S
Packaged with proteins: – 50S ribosome subunit ? 23S, 5S, 31 Proteins
– 30S ribosome subunit ? 16S, 21 Proteins ? 70S Ribosome
rRNAs in Eukaryotes (cytoplasmic) – 5S -18S – 5.8S -28S
Packaged with proteins: – 40S ribosome subunit ? 18S + 30 proteins – 60S ribosome subunit ? 5S, 5.8S, 28S + 45 proteins ? 80S Ribosome
六．tRNA processing 1.in prokaryotes
? Mature tRNAs are generated by processing longer pre-tRNA transcripts, which involves 1) specific exo- and endonucleolytic cleavage by RNases D, E, F and P (general) followed by 2) base modifications which are unique to each particular tRNA type. 2.in eukaryotes
? The pre-tRNA is synthesized with a 1) 16 nt 5’-leader, 2) a 14 nt intron and
3) two extra 3’-nucleotides.
4) CCA at the 3’-end (all tRNAs in both pro and euk)
七．RNase P
1.Ribonuclease P (RNase P) is an enzyme involved in tRNA processing that removes the 5' leader sequences from tRNA precursors, composed of one RNA and one protein molecule, simple RNP
2.RNase P enzymes are found in both prokaryotes and eukaryotes, being located in the nucleus of the latter where they are therefore small nuclear RNPs (snRNPs)
3.RNA component can catalyze pre-tRNA in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.
八．Ribozyme 1.Ribozyme (1)
① Ribozymes are catalytic RNA molecules that can catalyze particular biochemical reactions.
② RNase P RNA is a ribozyme. 2.Ribozyme (2)
? Self-splicing introns: the intervening RNA that catalyze the splicing of themselves from
their precursor RNA in the absence of proteins, and the joining of the exon sequences
? Self-cleaving RNA encoded by viral genome to cleave the concatameric molecules of the
viral genomic RNA into monomeric genome –sized lengths in some plant viruses Ribozymes can be used as therapeutic agents
九．Processing of mRNA
1.prokaryotes
① There is essentially no processing of prokaryotic mRNA, it can start to be translated before it has finished being transcribed.
② Prokaryotic mRNA is degraded rapidly from the 5’ end 2.eukaryotes
? In eukaryotes, mRNA is synthesized by RNA Pol II as longer precursors (pre-mRNA),
? Pre-mRNA molecules are processed to mature mRNAs by 5’-capping, 3’-cleavage and
polyadenylation, splicing and methylation. 3.Eukaryotic mRNA processing: overview
（一）Functions of the cap structure帽结构的功能 1. Helps prevent degradation 帮助防止降解
2. Helps transport into cytoplasm 帮助转运到细胞质中
3. Enhances translation / 增强转译 4. Helps remove the first intron 帮助去除第一个内含子 （二）Function of poly(A) tail ? Increased mRNA stability
? Increased translational efficiency ? Splicing of last intron
4.Introns and Exons / 内含子与外显子
? Introns: Sequences that do not code for protein and interrupt the coding regions. 内含子：不编码蛋白质并干涉打断编码区的序列。
? Exons: Parts of a gene that are expressed as protein. Exons are formed by the interruption of
coding regions by introns.
外显子：基因中被表达成蛋白质的部分。由内含子打断编码区域而形成。 5.Biochemical steps of pre-mRNA splicing
? Step 1: a cut is made at the 5′splice site, separating the left exon and the right intron-exon
molecule. The right intron-exon molecule forms a lariat, in which the 5′terminus of the intron becomes linked by a 5′-2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site
? Step 2: cutting at the 3′ splice site releases the free intron in lariat form, while the right
exon is ligated (spliced) to the left exon. 6.Spliceosome剪接体
? Catalyzes pre-mRNA splicing in nucleus
a. Composed of five snRNPs (U1, U2, U4, U5 and U6), other splicing factors, and the
pre-mRNA being assembled b. U1 binds to the 5’ splice site, then U2 to the branchpoint, then the tri-snRNP complex of U4,
U5 and U6. As a result, the intron is looped out and the 5’- and 3’ exon are brought into close proximity.
c. U2 and U6 snRNA are able to catalyze the splicing reaction. 7.Alternative splicing
? The generation of different mature mRNAs from a particular type of gene transcript can
occur by varying the use of 5’- and 3’- splice sites in four ways:
① ② ③ ④
By using different promoters By using different poly(A) sites By retaining certain introns
By retaining or removing certain exons
Section P
THE GENETIC CODE
AND tRNA: introduction to translation
一．The Protein Language
1.The Genetic Code
2.Anti-codons / 反密码子 ? 氨酰tRNA合成酶：
一种能够识别tRNA上的反密码子并将正确的氨基酸连接到其3’末端上去的酶。 ? 氨基酸是由氨酰tRNA合成酶加到tRNA上去的 3.tRNA secondary structure
The cloverleaf structure is a common secondary structural representation of tRNA molecules which shows the base paring of various regions to form four stems (arms) and three loops.
4.tRNA tertiary structure ? Formation:
9 hydrogen bones (tertiary hydrogen bones) to help the formation of tRNA tertiary structure, mainly involving in the base paring between the invariant bases.
? Hydrogen bonds:
Base pairing between residues in the D-and T-arms fold the tRNA molecule over into an L-shape, with the anticodon at one end and the amino acid acceptor site at the other. The base pairing is strengthened by base stacking interactions. 5.tRNA function
When charged by attachment of a specific amino acid to their 3’-end to become aminoacyl-tRNAs, tRNA molecules act as adaptor molecules in protein synthesis.
Section Q: PROTEIN SYNTHESIS
一．摇摆理论
1.Some highly purified tRNA molecules were found to interact with more than one codon, and this ability is correlated with the presence of modified nucleosides in the 5’-anticodon position, particularly inosine (formed by post-transcriptional processing of adenosine by anticodon deaminase which converts the 6-amino group to a keto group) 2.To explain the redundancy of the genetic code. 18 aa are encoded by more than one triplet codons which usually differ at 5’-anticodon base
3. 5'-anticodon base is able to undergo more movement than the other two bases and can thus form non-standard base pairs as long as the distances between the ribose units are close to normal (wobble rules).
二．The Wobble Rules（摇摆法则）
1.一种tRNA去识别不止一种密码子,这一增强了的碱基配对能力称为摇摆。 2.
3.Wobbled base pairing is to occur between the first position of the anticodon and the third position of the codon. If the first position of an anticodon is U, it will be able to pair with A or G at the third position of a codon. If the first position of an anticodon is G, it will be able to pair with C or U
4.Wobble pairing: non Waston-crick base paring
5.Modification to inosine allows pairing with C, U, and A. 6.One anticodon can be used to pair with 2 or 3 codons 一个反密码子可以用来与2~3个密码子配对
三．Mechanism of Translation in Prokaryotes 原核生物转译机理
1.SD sequence: translation start signal SD序列：翻译起始信号
SD序列：大肠杆菌中标明哪个AUG应该被用作起始密码子的共有序列。 ? Solely for prokaryotic translation
? A purine-rich sequence usually containing all or part of the sequence 5'-AGGAGGU-3' ? Upstream of the initiation codon in prokaryotic mRNA ? To position the ribosome for initiation of protein synthesis 2.Polysomes
① Each mRNA transcript is read simultaneously by more than one ribosome. ② A second, third, fourth, etc. ribosome starts to read the mRNA transcript before the first ribosome has completed the synthesis of one polypeptide chain.
③ Multiple ribosomes on a single mRNA transcript are called polyribosomes or polysomes.
④ Multiple ribosomes can not be positioned closer than 80 nt. 3.In prokaryotes, initiation requires
? the large and small ribosome subunits, ? the mRNA
? the initiator tRNA
? three initiation factors . 4.The three steps of elongation
① . aminoacyl-tRNA delivery Charged tRNA is delivered as a complex with EF-Tu and GTP .
② . peptide bond formation Peptidyl tranferase (50S ribosomal subunit) makes a peptide bond by joining the two adjacent amino acid without the input of more energy.
③ . translocation Translocase (EF-G), with the energy from GTP, moves the ribosome one codon along the mRNA, ejecting the uncharged tRNA and transferred the ribosome peptide from the mRNA. 5.Termination RF1 and RF2 Release factors and The release factors make recognizes the stop EF-G: remove the peptidyl transferase transfer codon with the help uncharged tRNA and the polypeptide to water, and release the mRNA,. of RF3 thus the protein is released
? RF1 recognizes UAA and UAG ? RF2 recognizes UAA and UGA
? RF3 helps the RF1 And RF2 to carry out the reaction ? The termination signal on A site : UAA.UAG,UGA
四.Translation (1/7)
1.To begin translation, a large and small ribosomal subunit, along with the initiating tRNA, assemble onto the mRNA.
2. In prokaryotes, the ribosome is correctly positioned
at the initiation codon by two RNA elements. Just upstream of the initiation codon is a Shine-Dalgarno sequence, which pairs with
the 3’ end of 16S rRNA.
3.Translation elongation
With the initiator tRNA placed at the P site of the ribosome, elongation can proceed. The next aminoacyl-tRNA enters the ribosome at the A site.
4.The amino acid at the P site is transferred to the tRNA at the A site. The ribosome moves one
codon farther along the mRNA, releasing the empty tRNA.
The A site is now free for the next incoming tRNA.
5.The cycle is repeated as the ribosome travels along the mRNA, resulting in a growing polypeptide chain.
6.Translation termination
Translation termination is triggered by a stop codon in the mRNA. Stop codons are recognized by release factors,
which help release the
fully-synthesized polypeptide chain from the ribosome.
7. Translation ends with dissociation of the ribosomal subunits.
五．Initiation in eukaryotes
1.Most of the differences in the mechanism of protein between prokaryotes and eukaryotes occur in the initiation stage, where a greater numbers of eIFs and a scanning process are involved in eukaryotes.
2.The eukaryotic initiator tRNA does not become N-formylated.
3.Initiation In contrast to the events in prokaryotes, initiation involves the initiation tRNA binding to the 40S subunits before it can bind to the mRNA. Phosphorylation of eIf2, which delivers the initiation tRNA, is an important control point. 4.Scanning
? There is no Shine-Delgarno (SD) sequence in eukaryotic mRNA, the mechanism of the
eukaryotic selecting the start codon must be different: Kozak proposed a scanning hypothesis
? Most eukaryotic mRNAs contain a short recognition sequence that greatly facilitate the initial
binding of mRNA to the small subunit of the ribosome. The consensus sequence for initiation of translation in vertebrates (also called Kozak sequence) is: Kozak sequences
5’ –CCRCCAUGG R=purine (G or A)
? To improve expression levels, it may be advantageous to design the cloned insert according
to Kozak's rules.
5.The scanning model / 扫描模型
6.Elongation The protein synthesis elongation cycle in prokaryotes and eukaryotes is quite similar.
The factors EF-Tu EF-Ts EF-G have direct eukaryotic equivalents called eEF1α eEF1βγ eEF2
7.Termination
Eukaryotes use only one release factors eRF, which requires GTP,recognize all three termination codons.
Termination codon is one of three (UAG, UAA, UGA) that causes protein synthesis to terminate.
8.Translational control
? In prokaryotes, the level of translation of different cistrons (顺反子)can be affected by: (a) the binding of short antisense molecules,
(b) the relative stability to nucleases of parts of the polycistronic mRNA , (c) the binding of proteins that prevent ribosome access.
? In eukaryotes (transcription control and mRNA processing) Translation level:
? protein binding can also mask the mRNA (masked mRNA) and prevent translation,
? repeats of the sequence 5'-AUUUA -3' (usually in the 3’-non coding region) can make the
mRNA unstable and less frequently translated. 9.Protein targeting
? The ultimate cellular location of proteins is often determined by specific, relatively short
amino acid sequence within the proteins themselves. These sequences can be responsible for proteins being secreted, imported into the nucleus or targeted to other organelles. ? Signal sequence, signal recognition particle (SRP) 10.Protein Targeting and Degradation
? Targeting mechanism involved a peptide signal sequence
? SRP (signal recognition particle) – move to ER then Gorgi lysosome, plasma membrane,
transport vesicles
? Amino-terminal sequence – mitochondria, chloroplast, bacterial export ? Internal signal sequence – nucleus proteins
? Degradation – ubiquitin-dependent proteolysis at proteasome
Eukaryotic protein targeting
英文分子生物学section M - Q复习知识点 - 图文
