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Nucleic acid (macro-molecules): Determining the correct order amino acids sequence → structure and One ribosomal RNA function Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira (DNA) contains all information → build the cells and tissues of • MOLECULAR organism Deoxyribonucleic acid (DNA) contains the BIOLOGY information prescribing the sequence of • SIXTH EDITION This information is arranged in units termed • CHAPTER 4 Ribonucleic acid (RNA) serves in the cellular machinery that chooses and links amino acids RNA + + → in the correct sequence • Basic Molecular Genetic ribosomal ribonucleoprotein The central dogma: DNA ⌫ RNA ⌫ Protein complexes (rRNPs) Mechanisms DNA and RNA are polymers of nucleotide ©Copyright 2008 W. 2008 H. © FreemanW. H. Freeman and and Company Company subunits

Chemical structure of the principal bases (ch3)

Monomers→polymers DNA: ATCG RNA: AUCG

專有名詞要不止要 Four basic molecular genetic processes: 放在心中,更要放 Protein synthesis: 1 to 3 在腦中 rRNA: ribosomal RNA; tRNA: transfer RNA rNTPS: ribonucleoside triphosphate monomers; dNTP:deoxyribonucleoside triphophate Structure of nucleic acid A nucleic acid strand is a linear polymer with end to end directionality

REMEMBER:

DNA = deoxyribonucleotides; RNA = ribonucleotides (OH-groups at the 2’ position) Note the directionality of DNA (5’-3’ & 3’- 5’) or RNA (5’-3’) DNA = A, G, C, T ; RNA = A, G, C, U

Nucleotide subunits are linked together by phosphodiester bonds

Native DNA is a double helix of complementary antiparallel Nucleic acid as hetero-polymers strands 1953, Watson and Francis: proposed Nucleosides, nucleotides DNA and RNA strands that DNA has a double-helical structure Nature, 4356, 737-728 (1953) DNA consists of two associated polynucleotide strands that wind together to form a double helix. 5’→3’; 3’→5’ antiparallel : H-bond formation, A-T (2) and G-C (3) (Ribose sugar, (2’-deoxy ribose sugar, Complementary: two polynucleotide RNA precursor) DNA precursor) consequence of the size, shape and chemical composition, by base pair interaction (A-T and C-G)

There are two major forces that contribute to stability of helix formation: Hydrogen bonding in base-pairing Hydrophobic interactions in base (2’-deoxy thymidine tri- stacking (堆) phosphate, nucleotide) So … Most DNA in cells is a right handed DNA RNA helix X-ray data of DNA: (B-form) 1.The stacked bases are regularly spaced 0.34-0.36nm 2.Helix makes a complete turn every 3.6nm, about 10.5 pairs per turn.

B-DNA

A-DNA

B DNA most common d(CGCGAATTCGCG)•d(CGCGAATTCGCG) A DNA, in low humidity condition, B transform to A form; RNA-RNA, Z-DNA RNA-DNA d(AGCTTGCCTTGAG)•d(CTCAAGGCAAGCT) Z DNA, short DNA molecules composed of alternating purine-pyrimidine nucleotides (GC), right transform to left d(CGCGCGCGCGCG)•d(CGCGCGCGCGCG) DNA compositional biases B-DNA A-DNA Base compositions of genomes: G+C (and therefore also A+T) content varies between different genomes

The GC-content is sometimes used to classify organism in R.H. helix R.H. helix taxonomy

High G+C content bacteria: Actinobacteria e.g. in Streptomyces coelicolor it is 72% 鏈黴菌 Low G+C content: Plasmodium falciparum (~20%) 瘧原虫 Z-DNA Other examples:

Saccharomyces cerevisiae (yeast) 38% Arabidopsis thaliana (plant) 36% Escherichia coli (bacteria) 50% L.H. helix

TBP protein can binds to Why RNA degradation more easy than DNA??? the minor groove of specific DNA RNA DNA (rich AT)→ untwisting and sharply bending the double helix → transcription ability ↑

Why is rich AT region ? Base-catalyzed hydrolysis of RNA

2’-OH site as a nucleophile at normal pH → attacking phophodiester bond→ degradation

In 2’ site, DNA is more stable than RNA.

DNA can undergo reversible strand separation (denaturation) SV40 viral DNA

Many prokaryotic genomic DNA and viral DNA are circular molecules. Circular DNA molecules in eukaryotic mitochondria and chloroplasts Circular DNA without end, when replication: open DNA → unwinding DNA → torsional (扭 力) stress → winding (纏繞) → formed super-coil (超螺旋) Topoisomerase I (bacterial and eukaryotic cell has) → bind to DNA → breaks a phosphodiester bond in one strands DNA formed nick → loss Tm: melting temperature supercoiled → ligates the two ends of the broken strand. G-C more → need more energy Topoisomerase II, breaks two strands DNA Denature of single stranded DNA → random coil (without organized structure) Renature vs hybribization Supercoils Supercoiling of DNA can only occur in closed-circular DNA or linear DNA where Supercoiling induced by separating the strands the ends are fixed. of duplex DNA (eg., during DNA replication)

Underwinding produces negative supercoils, DNA (double strain) → open → single strain → replication or transcription→ whereas overwinding produces positive supercoils. spuercoiling → need topoisomerase

Transcription of protein-coding genes and formation of functional mRNA

Relaxed and supercoiled plasmid DNA → RNA → Protein → function

ATCG AUCG AA

mRNA protein –coding gene tRNA rRNA

Encode: gene → mRNA → protein

DNA replication Direction 5’ to 3’ ~800 nd/sec RNA polymeration: Direction 5’ to 3’ ~40 nd/sec Direction 5’ to 3’ ~15 aa/sec Different types of RNA exhibit various conformations related Three Different Classes of RNA their functions AUCG: CG has 3 H-bond 1) rRNA (ribosomal) Most RNA are single strand Various RNA → carry out specific • large (long) RNA molecules functions • structural and functional components of Eukaryotic cell, RNA self-splicing > Secondary structure H-bond • highly abundant dependent 2) mRNA (messenger) 5-10 nucleotides >10 nucleotides • typically small (short) • encode proteins • multiple types, not abundant 3) tRNA (transfer) and small ribosomal • very small • Important in translation

Not all genes encode proteins

transcription DNA RNA

DNA RNA A template DNA strand is transcribed into a complementary RNA chain by RNA. Deoxyribonucleic acid Ribonucleic acid Ribonucleoside triphosphate (rNTP) are polymerized to form a complementary RNA by RNA polymerase. Polymerization involves a nucleophilic attack by the 3’ ATCG AUCG oxygen in the growing RNA chain on the a phosphate of the next nucleotide → formed More rigid More flexible phosphodiester bond and release pyrophosphate More stable More unstable Direction: 5’→ 3’; opposite in polarity to their template DNA strands mRNA, rRNA, tRNA DNA A→U T→A C→ G G→C transcribed to RNA

Release PPi The micro RNA (miRNA): 1. Regulate specific mRNA 2. Produced by RNA polymerase RNA polymerase begins transcription is +1 Downstream: +, Upstream: -

pri-miRNA ：由基因組中轉錄出來 Drosha ：一種RNaseIII pre-miRNA：由Drosha切割pri-miRNA 而來 Exportin-5 ：可將pre-miRNA由細胞核運到 細胞質。 miRNA duplex ： pre-miRNA被切割後的產物 (20~22個鹼基 ) mature miRNA：有活性的單鏈miRNA

Bacterial (Prokaryotic) Transcription Three stages in transcription

Promoters - DNA sequences that guide RNA polymerase to the beginning of a gene (transcription initiation site). Terminators - DNA sequences that specify then termination of RNA synthesis Many Need transcription factor help binding for help RNA and release of RNAP from the DNA. polymerase binding RNA Polymerase (RNAP) - Enzyme for synthesis of RNA. Reaction (ordered series of steps) 1) Initiation. 2) Elongation. About 14 base pairs 3) Termination. Recognition

rNTP vs.dNTP Three stages in transcription Termination of transcription Two mechanisms 1) Rho - the termination factor protein 初期 – rho is an ATP-dependent helicase – it moves along the RNA transcript, finds the "bubble", About 8 base pair For continuous RNA synthesis and unwinds it and releases the RNA chain. without dissociation

2) Rho-Independent - termination sites in DNA

inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript

Rho-Dependent Transcription Termination (depends on a protein AND a DNA sequence) Termination of transcription

Two mechanisms G/C -rich site 2) Rho-Independent - termination sites in DNA – inverted repeat, rich in G:C, which forms a RNAP slows down stem-loop in RNA transcript

Rho helicase catches up

Elongating complex is disrupted Rho-independent Rho-Independent Transcription Termination transcription (depends on DNA sequence - NOT a protein factor) termination

• RNAP pauses when it reaches a termination site. • The pause may give the hairpin structure time to fold • The fold disrupts important interactions between the RNAP and its RNA product • The U-rich RNA can dissociate from the template Stem-loop structure DNA RNA A T • The complex is now disrupted T U and elongation is terminated G C C G

Transcriptional mechanism-1

Bacterial RNA polymerase Structure of RNA polymerase RNA Polymerase • RNA Polymerase is a spectacular (壯觀) enzyme, functioning in: 1 Recognition of the region RNA polymerase are similar in eukaryotic and prokaryotic cell 2 Melting of DNA (Helicase + Topisomerase); unwinding DNA Five subunit: 3 RNA Priming (Primase) 2 large subunit: β, β’; 2 smaller subunits 4 RNA Polymerization; add rNTP α and ω (only Stabilizes and assembly 5 Recognition of sequence of its subunits) Only a single RNA polymerase RNA-DNA hybrid Length? 3 to 9 bases, it is short and transit (prokaryotic) In E.coli, RNA polymerase is 465 kD In Bacterial which can hold~16 bp complex, with 2 α, 1 β, 1 β', 1 σ In yeast which can hold ~25 bp β' binds DNA β binds rNTPs and interacts withMg σ 2+ β and β ' together make up the active site α subunits appear to be essential for assembly and for activation of Thus, RNAP is a multisubunit enzyme enzyme by regulatory proteins Current model of bacterial RNA polymerase bond to a promoter

Different Types of RNA Polymerase One model for transcriptional activation

In Bacteria (simple system) - all three classes are transcribed by the same RNA polymerase (RNAP for short)

In (complex system) - each class is transcribed by a different RNA Polymerase •RNAP I - rRNAs •RNAP II - mRNAs •RNAP III - tRNAs & small ribosomal RNAs

•Remember: only RNAP did not transcript !!!! Need many transcription factor (protein)

Flash-2 Gene Regulation Organization of genes differs in prokaryotic and eukaryotic DNA Protein complex → DNA → open/tight DNA → transcription Genomes In prokaryotic: Transcription is regulated by proteins binding to or near 1. logic: genes devoted (致力於) to a promoters single metabolic goal; protein synthesis – Three types of proteins involved: from a contiguous array in DNA. It • Specificity factors means that one gene → one protien→. • 可以多段有功能基因連在一起 one → one goal (function) • Activators 2. Arrangement of genes in a functional – Repressors: bind to specific sites on DNA group is cell an operon, because it • Called operators operate as a unit from a single • Either near or overlapping the promoter promoter. One promoter → one gene ( • Block movement of RNA-polymerase or genes) → one protein (or proteins) -Activators: bind to specific sites on DNA, help RNAP moving 3. The genes are closely packed with very few non-coding gaps Operon: arrangement of genes in a functional group DNA → direct to co-linear mRNA → → translated protein

Eukaryotic precursor mRNA are processed to form functional mRNAs In prokaryotic, protein synthesis can occur in 5’ or 3’ In eukaryotic end of mRNA; transcription and translation can 1. RNA → discontinuous in occur at the same time. corresponding DNA sequence In eukaryotic, in nucleus DNA → transcription → 2. DNA contain (coding precursor mRNA → procession → functional sequence) and (non- mRNA → transport to → translated to protein-coding segments) protein; Transcription and translation are in 3. DNA → RNA, remove introns and different time and place. carefully stitched back together to Pre-mRNA are modified at the tow ends, and keep in produced many mRNAs mRNA. It can protect the degradation of RNA form 4. Functional (mature) mRNA from nucleus to cytoplasm. Don’t need DNA template. precursor mRNA processed Modification of 5’ end: by RNA polymerase II → add (splicing) 5’cap; methylation 5. DNA → pre-mRNA → splicing → Modification of 3’ end: by poly A polymerase, add mature mRNA→ protein → add 100-250 A and produced poly A tail. modification → mature protein mRNA processing – RNA splicing, 5’ and 3’ retain noncoding regions (untranslated regions; UTRs). In mammalian mRNA, 5’ UTR about >100 Each gene is transcripbed from its own promoter nucleotides, 3’ UTR about several kilobases Tryptophan (trp) Tryptophan metabolite enzyme The ribose of the second nucleotide also is methylated Alternative RNA splicing increase the number of proteins expressed from a single eukaryotic gene RNA Processing: • : transcription and translation can be One gene → RNA splicing → different RNA→ different protein concurrent. Isoform: by alternative splicing production of different forms of a • Eukaryotes: Nucleus (RNA synthesis) and cytoplasm protein. (Protein synthesis) are separated. One gene can lead to more than one protein (e.q. antibodies) • undergoes several modifications. 7 Exons: part of the gene that is expressed. • 5’ cap is added to 5’ nucleotide; m Gppp (Stability) Introns: part of gene that is spliced out • String of adenylic acids are added to the 3’ end (Poly from pre-mRNA. A tail) Sometimes some exons are also spliced out. • Splicing: internal cleavage to excise introns followed by ligation of coding exons Formed three protein-coding

5’ and 3’ ends of eukaryotic mRNA Functions of 5’ cap and 3’ polyA

Both cap and polyA contribute to stability of mRNA: – Most mRNAs without a cap or polyA are degraded rapidly. – Shortening of the polyA tail and decapping are part of one pathway for RNA degradation in yeast. Need 5’ cap for efficient translation: Add a GMP. Cut the pre-mRNA – Eukaryotic translation initiation factor 4 (eIF4) Methylate it and and add A’s recognizes and binds to the cap as part of initiation. 1st few nucleotides – Assists mRNA export to the cytoplasm 轉到ch6 p216 The structure of genes and chromosomes

p249

Gene: DNA regions encoding proteins or functional RNA : non-functional DNA, non-coding regions of DNA Extron: functional DNA, of DNA Transposable (mobile) DNA: non-coding region, repeat, evolutionary DNA must be contend: human cell has 2 meters DNA!!!!!SO must be highly compacted In eukaryotes, DNA + protein → chromatin → chromosome histone Homologous recombination and generate genetic diversity Eukaryotic gene structure A gene: as the entire nucleic acid sequence that is necessary for the synthesis of a functional gene product Coding region: coding amino acids sequence, or functional RNA : transcript regions, not coding region; it regulated transcriptive activity Most eukaryotic genes contain introns and produce mRNA encoding single proteins Generate genetic diversity among the individuals of Simple and complex transcriptions units are found in eukaryotic cells a species by causing the Cistron: a genetic unit encoding a single polypeptide exchange of large regions Polycistron: a genetic unit (not a only a gene) encoding multiple of chromosomes between polypeptides; also called operon, like prokaryotic cell for live the maternal and paternal Most eukaryotic cell has mono-cistron. pair of homologous chromosomes during the Prokaryotes have compact genomes and their transcripts often contain cellular division the multiple protein coding regions (called open reading frames or ORFs) generates germ cells

These mRNAs are called polycistronic mRNAs (a cistron is a concept that is similar to a gene, and for many genes the cistron=gene)

Homologous recombination: meiosis

Exon 3 is lost

L: non-coding repeat, also called Transposable (mobile) DNA its easy to homologous recombination Simple and complex eukaryotic transcription

For gene that are transcribed from different promoters (regulator Mutation control region: no mRNA expression → no protein → no function factor) in different cell type Mutation Exon : mRNA expression (some wrong) → abnormal protein → activity change

Protein-coding genes may be solitary or belong to a gene family

Solitary gene: in , 20-50% protein coding gene are reprsented Duplicated gene: gene family → protein family homologous duplicated gene encode protein with similar New Roles of RNA

RNAi - RNA interference siRNA- active molecules in RNA interference; degrades mRNA (act where they originate) miRNAs - tiny 21–24-nucleotide RNAs; probably acting as translational regulators of protein-coding mRNAs

stRNA - Small temporal RNA; (ex. lin-4 and let-7 in Caenorhabditis elegans snRNA - Small nuclear RNA; includes spliceosomal RNAs (processing) snoRNA - Small nucleolar RNA; most known snoRNAs are involved in rRNA modification

Alternative RNA splicing increases the number or proteins expressed from a single eukaryotic gene Production of heavy chain genes in mouse by recombination of V, D, J, and C gene segments during development Higher have multidomain tertiary structure only from a small number of exons. • Alternative splicing Single gene →Multiple introns→alternative splicing → protein isoforms – Different mRNAs Alternative splicing: The presence of multiple can be produced by introns in many eukaryotic genes permits same transcript expression of multiple, related proteins form a single gene. > 20 isoforms fibronectin from different alternatively spliced mRNA

Cell type specific splicing of fibronectin pre-mRNA Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Differences Between Transcription In Prokaryotes and Eukaryotes

Transcription And Translation In ------the same time Eukaryotic Transcription and translation------different time

Processing Eukaryotic mRNA