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Replication and gene exression

DNA replication and gene expression are the two main processes by which DNA is copied and expressed. DNA replication involves unwinding the parental DNA strands, synthesizing new complementary strands according to base pairing rules, resulting in two identical copies of the original DNA. Gene expression involves transcription of DNA into RNA and translation of RNA into protein. Transcription occurs in the nucleus and involves RNA polymerase making an RNA copy of a gene. The RNA then undergoes processing before being exported to the cytoplasm for translation by ribosomes into a polypeptide chain according to the genetic code.

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DNA replication and gene expression C T A A T CG GC A C G AT AT A T TA C TA 0.34 nm 3.4 nm (a) Key features of DNA structure G 1 nm G (c) Space-filling model T Rahna K Rathnan Assistant Professor Sahrdaya college of engineering and Technology
DNA replication • The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parentalstrand and one new strand. A C T A G A C T A G A C T A G A C T A G T G A T C T G A T C A C T A G A C T A G T G A T C T G A T C T G A T C T G A T C
DNA Replication is “Semi-conservative” • Newly synthesised duplex DNA conserve one strand of parent DNA and other strand will be newly synthesised • One original strand was used as a template to make the new strand
DNA Replication • Require – Template DNA – More than dozen enzymes and proteins • DNA synthesis enzyme : DNA polymerase • DNA synthesis direction ---- 5’ to 3’ • The replication of a DNA molecule begins at special sites called origins of replication • the two strands are separated
Origins of Replication• A eukaryotic chromosome may have hundreds or even thousands of replication origins Replication begins at specific sites where the two parental strands separate and form replication bubbles. The bubbles expand laterally, as DNA replication proceeds in both directions. the replication bubbles fuse, and synthesis of the daughter strands is complete. 1 2 3 Origin of replication Bubble Parental (template) strand Daughter (new) strand Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of (b)(a) 0.25 µm
Mechanism of DNA Replication • DNA replication is catalyzed by DNA polymerase – needs an RNA primer • RNA primase synthesizes primer on DNA strand • DNA polymerase adds nucleotides to the 3’ end of the growing strand
Mechanism of DNA Replication • Nucleotides are added by complementary base pairing with the template strand • The substrates, deoxyribonucleoside triphosphates, are hydrolyzed and releasing energy for DNA synthesis.
The Mechanism of DNA Replication • DNA synthesis on the leading strand is continuous • The lagging strand grows the same general direction as the leading strand (in the same direction as the Replication Fork). However, DNA is made in the 5’-to-3’ direction • DNA synthesis on the lagging strand is discontinuous • DNA is added as short fragments (Okasaki fragments) – subsequently ligated together
DNA polymerase I degrades the RNA primer and replaces it with DNA
The Mechanism of DNA Replication • DNA helicases unwind the double helix • Single-stranded DNA binding proteins stabilize the SS DNA • RNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added. • DNA polymerase III extends the strand in the 5’-to- 3’ direction • DNA polymerase I degrades the RNA primer and replaces it with DNA • DNA ligase joins the DNA fragments into a continuous daughter strand
Enzymes in DNA replication Helicase unwinds parental double helix Binding proteins stabilize separate strands DNA polymerase III binds nucleotides to form new strands Ligase joins Okazaki fragments and seals other nicks in sugar- phosphate backbone Primase adds short primer to template strand DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases
• Helicase protein binds to DNA sequences called origins and unwinds DNA strands. • Single strand Binding proteins prevent single strands from rewinding. • Primase protein makes a short segment of RNA complementary to the DNA, a primer. 5’ 3’ 5’ 3’ 3’5’ 5’3’ Replication
Overall direction of replication 5’3’ 5’ 3’ 5’ 3’ 3’5’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer. Replication
• DNA polymerase enzyme adds DNA nucleotides to the RNA primer. • DNA polymerase proofreads bases added and replaces incorrect nucleotides. 5’ 5’ Overall direction of replication 5’ 3’ 5’ 3’ 3’ 3’ Replication
5’ 5’3’ 5’ 3’ 3’ 5’ 3’ Overall direction of replication • Leading strand synthesis continues in a 5’ to 3’ direction. Replication
3’5’ 5’ 5’3’ 5’ 3’ 3’ 5’ 3’ Overall direction of replication Okazaki fragment • Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication
5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication
3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 5’3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication
5’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ • Exonuclease activity of DNA polymerase I removes RNA primers. Replication
Polymerase activity of DNA polymerase I fills the gaps. Ligase forms bonds between sugar-phosphate backbone. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ Replication
Replication Fork Overview
Gene Expression • Synthesis of protein from DNA through RNA • The process by which DNA directs protein synthesis, gene expression includes two stages – transcription – translation
Transcription and Translation • Cells are governed by a cellular chain of command – DNA → RNA → protein • Transcription – Is the synthesis of RNA under the direction of DNA – Produces messenger RNA (mRNA) • Translation – Is the actual synthesis of a polypeptide – occurs under the direction of mRNA – Occurs on ribosomes
Transcription and Translation • In a eukaryotic cell the nuclear envelope separates transcription from translation • Extensive RNA processing occurs in the nucleus Eukaryotic cell. The nucleus provides a separate compartment for transcription. The original RNA transcript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. (b) TRANSCRIPTION RNA PROCESSING TRANSLATION mRNA DNA Pre-mRNA Polypeptide Ribosome Nuclear envelope
Transcription • Transcription is the DNA-directed synthesis of RNA • RNA synthesis – Is catalyzed by RNA polymerase – uracil substitutes for thymine
RNA • RNA is single stranded, not double stranded like DNA • RNA is short, only 1 gene long, where DNA is very long and contains many genes • RNA uses the sugar ribose instead of deoxyribose in DNA • RNA uses the base uracil (U) instead of thymine (T) in DNA. Table 17.1
Synthesis of an RNA Transcript • The stages of transcription are – Initiation – Elongation – Termination Promoter Transcription unit RNA polymerase Start point 5′ 3′ 3′ 5′ 3′ 5′ 5′ 3′ 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ 5′ 5′ Rewound RNA R N A tr a ns cri pt 3′ 3′ Completed RNA transcript Unwound DNA RNA transcript Template strand of DNA DNA 1 Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. 2 Elongation. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5′ → 3 ′. In the wake of transcription, the DNA strands re-form a double helix. 3 Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA.
• Promoters signal the initiation of RNA synthesis • Transcription factors help eukaryotic RNA polymerase recognize promoter sequences TRANSCRIPTION RNA PROCESSING TRANSLATION DNA Pre-mRNA mRNA Ribosome Polypeptide T A T AAA A ATAT T T T TATA box Start point Template DNA strand 5′ 3′ 3′ 5′ Transcription factors 5′ 3′ 3′ 5′ Promoter 5′ 3′ 3′ 5′5′ RNA polymerase II Transcription factors RNA transcript Transcription initiation complex Eukaryotic promoters1 Several transcription factors 2 Additional transcription factors 3 Synthesis of an RNA Transcript - Initiation
Synthesis of an RNA Transcript - Elongation • RNA polymerase synthesizes a single strand of RNA against the DNA template strand (anti-sense strand), adding nucleotides to the 3’ end of the RNA chain • As RNA polymerase moves along the DNA it continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides Elongation RNA polymerase Non-template strand of DNA RNA nucleotides 3′ end C A E G C A A U T A G G T T A A C G U A T C A T C C A A T T G G 3′ 5′ 5′ Newly made RNA Direction of transcription (“downstream”) Template strand of DNA
• Specific sequences in the DNA signal termination of transcription • When one of these is encountered by the polymerase, the RNA transcript is released from the DNA and the double helix can zip up again. Synthesis of an RNA Transcript - Termination
Transcription Overview
• Most eukaryotic mRNAs aren’t ready to be translated into protein directly after being transcribed from DNA. • mRNA requires processing. • Transcription of RNA processing occur in the nucleus. – the messenger RNA moves to the cytoplasm for translation. • The cell adds a protective cap to one end, and a tail of A’s to the other end. • These both function to protect the RNA from enzymes that would degrade Post Termination RNA Processing
• Most of the genome consists of non-coding regions called introns – Non-coding regions may have specific chromosomal functions or have regulatory purposes – Introns also allow for alternative RNA splicing • Thus, an RNA copy of a gene is converted into messenger RNA by doing 2 things: – Add protective bases to the ends – Cut out the introns Post Termination RNA Processing
Alteration of mRNA Ends • Each end of a pre-mRNA molecule is modified in a particular way – The 5′ end receives a modified nucleotide cap – The 3′ end gets a poly-A tail A modified guanine nucleotide added to the 5′ end 50 to 250 adenine nucleotides added to the 3′ end Protein-coding segment Polyadenylation signal Poly-A tail3′ UTR Stop codonStart codon 5′ Cap 5′ UTR AAUAAA AAA…AAA TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide G P P P 5′ 3′
RNA Processing - Splicing • The original transcript from the DNA is called pre-mRNA. • It contains transcripts of both introns and exons. • The introns are removed by a process called splicing to produce messenger RNA (mRNA)
RNA Processing • Proteins often have a modular architecture consisting of discrete structural and functional regions called domains • In many cases different exons code for the different domains in a protein Figure 17.12 Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 1 Domain 2 Polypeptide
Translation • Translation is the RNA-directed synthesis of a polypeptide • Translation involves – mRNA – Ribosomes - Ribosomal RNA – Transfer RNA – Genetic coding - codons TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Polypeptide Amino acids tRNA with amino acid attachedRibosome tRNA Anticodon mRNA Trp Phe Gly A G C A A A C C G U G G U U U G G C Codons5′ 3′
The Genetic Code • Genetic information is encoded as a sequence of nonoverlapping base triplets, or codons DNA molecule Gene 1 Gene 2 Gene 3 DNA strand (template) TRANSCRIPTION mRNA Protein TRANSLATION Amino acid A C C A A A C C G A G T U G G U U U G G C U C A Trp Phe Gly Ser Codon 3′ 5′ 3′5′
The Genetic Code • Codons: 3 base code for the production of a specific amino acid, • Present on mRNA • there are 64 possible codons • 64 codons but only 20 amino acids, therefore most have more than 1 codon • 3 of the 64 codons are used as STOP signals – they are found at the end of every gene and mark the end of the protein • One codon is used as a START signal: it is at the start of every protein • Universal: in all living organisms
The Genetic Code • A codon in messenger RNA is either translated into an amino acid or serves as a translational start/stop signal Second mRNA base U C A G U C A G UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Met or start Phe Leu Leu lle Val UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala UAU UAC UGU UGC Tyr Cys CAU CAC CAA CAG CGU CGC CGA CGG AAU AAC AAA AAG AGU AGC AGA AGG GAU GAC GAA GAG GGU GGC GGA GGG UGG UAA UAG Stop Stop UGA Stop Trp His Gln Asn Lys Asp Arg Ser Arg Gly U C A G U C A G U C A G U C A G FirstmRNAbase(5′end) ThirdmRNAbase(3′end) Glu
Transfer RNA • Consists of a single RNA strand that is only about 80 nucleotides long • Each carries a specific amino acid on one end and has an anticodon on the other end • A special group of enzymes pairs up the proper tRNA molecules with their corresponding amino acids. • tRNA brings the amino acids to the ribosomes Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3′ end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.) (a) 3′ C C A C G C U U A A GACACCU * G C * * G U G U *CU * G AG G U * *A * A A G U C A G A C C * C G A G A G G G * * GA CUC*AU U U A G G C G 5′ Amino acid attachment site Hydrogen bonds Anticodon A The “anticodon” is the 3 RNA bases that matches the 3 bases of the codon on the mRNA molecule
Ribosomes • Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis • The 2 ribosomal subunits are constructed of proteins and RNA molecules named ribosomal RNA or rRNA
Composition of whole ribosomes and of ribosomal subunits in mammalian cells
Building a Polypeptide • divide translation into three stages – Initiation – Elongation – Termination
Building a Polypeptide • The AUG start codon is recognized by methionyl-tRNA or Met • Once the start codon has been identified, the ribosome incorporates amino acids into a polypeptide chain • RNA is decoded by tRNA (transfer RNA) molecules, which each transport specific amino acids to the growing chain • Translation ends when a stop codon (UAA, UAG, UGA) is reached
• Translation starts (primarily) at AUG • AUG codes for methionine • 16S rRNA of the small 30S ribosomal subunit recognizes the ribosomal binding site on the mRNA -Shine-Dalgarno sequence or SD • Shine-Dalgarno sequence is complementary to sequence on 16S rRNA. • SD helps to correctly position the ribosome onto the mRNA so that the P site is directly on the AUG initiation codon INITIATION
Initiation of Translation • The initiation stage of translation brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome Large ribosomal subunit The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiation factors (not shown) are required to bring all the translation components together. GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid. 2 Initiator tRNA mRNA mRNA binding site Small ribosomal subunit Translation initiation complex P site GDPGTP Start codon A small ribosomal subunit binds to a molecule of mRNA. In a prokaryotic cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, with the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine (Met). 1 Met Met U A C A U G E A 3′ 5′ 5′ 3′ 3′5′ 3′5′
Amino end Growing polypeptide Next amino acid to be added to polypeptide chain tRNA mRNA Codons 3′ 5′ Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base- pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. (c) Building a Polypeptide
Elongation of the Polypeptide Chain • In the elongation stage, amino acids are added one by one to the preceding amino acid Amino end of polypeptide mRNA Ribosome ready for next aminoacyl tRNA E P A E P A E P A E P A GDP GTP GTP GDP 2 2 site site5′ 3′ TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Codon recognition. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. 1 Peptide bond formation. An rRNA molecule of the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site. 2 Translocation. The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site. 3
Termination of Translation • The final stage is termination when the ribosome reaches a stop codon in the mRNA Release factor Free polypeptide Stop codon (UAG, UAA, or UGA) 5′ 3′ 3′ 5′ 3′ 5′ When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. 1 The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. 2 3 The two ribosomal subunits and the other components of the assembly dissociate.
Translation • The final step in translation is termination. When the ribosome reaches a STOP codon, there is no corresponding transfer RNA. • Instead, a small protein called a “release factor” attaches to the stop codon. • The release factor causes the whole complex to fall apart: messenger RNA, the two ribosome subunits, the new polypeptide. • The messenger RNA can be translated many times, to
A summary of transcription and translation in a eukaryotic cell Figure 17.26 TRANSCRIPTION RNA is transcribed from a DNA template. DNA RNA polymerase RNA transcript RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. Exon Poly-A RNA transcript (pre-mRNA) Intron NUCLEUS Cap FORMATION OF INITIATION COMPLEX After leaving the nucleus, mRNA attaches to the ribosome. CYTOPLASM mRNA Poly-A Growing polypeptide Ribosomal subunits Cap Aminoacyl-tRNA synthetase Amino acid tRNA AMINO ACID ACTIVATION Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Activated amino acid TRANSLATION A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) Anticodon A C C A A A U G G U U U A U G U A CE A Ribosome 1 Poly-A 5′ 5′ 3′ Codon 2 3 4 5
Post-translation • The new polypeptide is now floating loose in the cytoplasm if translated by a free ribosme. • It might also be inserted into a membrane, if translated by a ribosome bound to the endoplasmic reticulum. • Polypeptides fold spontaneously into their active configuration, and they spontaneously join with other polypeptides to form the final proteins. • Sometimes other molecules are also attached to the polypeptides: sugars, lipids, phosphates, etc. All of these have special purposes for protein function.

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Replication and gene exression

  • 1.
    DNA replication andgene expression C T A A T CG GC A C G AT AT A T TA C TA 0.34 nm 3.4 nm (a) Key features of DNA structure G 1 nm G (c) Space-filling model T Rahna K Rathnan Assistant Professor Sahrdaya college of engineering and Technology
  • 2.
    DNA replication • Theparent molecule unwinds, and two new daughter strands are built based on base-pairing rules (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parentalstrand and one new strand. A C T A G A C T A G A C T A G A C T A G T G A T C T G A T C A C T A G A C T A G T G A T C T G A T C T G A T C T G A T C
  • 3.
    DNA Replication is“Semi-conservative” • Newly synthesised duplex DNA conserve one strand of parent DNA and other strand will be newly synthesised • One original strand was used as a template to make the new strand
  • 4.
    DNA Replication • Require –Template DNA – More than dozen enzymes and proteins • DNA synthesis enzyme : DNA polymerase • DNA synthesis direction ---- 5’ to 3’ • The replication of a DNA molecule begins at special sites called origins of replication • the two strands are separated
  • 5.
    Origins of Replication•A eukaryotic chromosome may have hundreds or even thousands of replication origins Replication begins at specific sites where the two parental strands separate and form replication bubbles. The bubbles expand laterally, as DNA replication proceeds in both directions. the replication bubbles fuse, and synthesis of the daughter strands is complete. 1 2 3 Origin of replication Bubble Parental (template) strand Daughter (new) strand Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of (b)(a) 0.25 µm
  • 6.
    Mechanism of DNAReplication • DNA replication is catalyzed by DNA polymerase – needs an RNA primer • RNA primase synthesizes primer on DNA strand • DNA polymerase adds nucleotides to the 3’ end of the growing strand
  • 7.
    Mechanism of DNAReplication • Nucleotides are added by complementary base pairing with the template strand • The substrates, deoxyribonucleoside triphosphates, are hydrolyzed and releasing energy for DNA synthesis.
  • 8.
    The Mechanism ofDNA Replication • DNA synthesis on the leading strand is continuous • The lagging strand grows the same general direction as the leading strand (in the same direction as the Replication Fork). However, DNA is made in the 5’-to-3’ direction • DNA synthesis on the lagging strand is discontinuous • DNA is added as short fragments (Okasaki fragments) – subsequently ligated together
  • 10.
    DNA polymerase Idegrades the RNA primer and replaces it with DNA
  • 11.
    The Mechanism ofDNA Replication • DNA helicases unwind the double helix • Single-stranded DNA binding proteins stabilize the SS DNA • RNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added. • DNA polymerase III extends the strand in the 5’-to- 3’ direction • DNA polymerase I degrades the RNA primer and replaces it with DNA • DNA ligase joins the DNA fragments into a continuous daughter strand
  • 12.
    Enzymes in DNAreplication Helicase unwinds parental double helix Binding proteins stabilize separate strands DNA polymerase III binds nucleotides to form new strands Ligase joins Okazaki fragments and seals other nicks in sugar- phosphate backbone Primase adds short primer to template strand DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases
  • 13.
    • Helicase proteinbinds to DNA sequences called origins and unwinds DNA strands. • Single strand Binding proteins prevent single strands from rewinding. • Primase protein makes a short segment of RNA complementary to the DNA, a primer. 5’ 3’ 5’ 3’ 3’5’ 5’3’ Replication
  • 14.
    Overall direction of replication 5’3’ 5’ 3’ 5’ 3’ 3’5’ DNApolymerase enzyme adds DNA nucleotides to the RNA primer. Replication
  • 15.
    • DNA polymeraseenzyme adds DNA nucleotides to the RNA primer. • DNA polymerase proofreads bases added and replaces incorrect nucleotides. 5’ 5’ Overall direction of replication 5’ 3’ 5’ 3’ 3’ 3’ Replication
  • 16.
    5’ 5’3’ 5’ 3’ 3’ 5’ 3’ Overall direction of replication •Leading strand synthesis continues in a 5’ to 3’ direction. Replication
  • 17.
    3’5’ 5’ 5’3’ 5’ 3’ 3’ 5’ 3’ Overall direction ofreplication Okazaki fragment • Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication
  • 18.
    5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’3’ Leadingstrand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication
  • 19.
    3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 5’3’ Leadingstrand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication
  • 20.
    5’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ •Exonuclease activity of DNA polymerase I removes RNA primers. Replication
  • 21.
    Polymerase activity ofDNA polymerase I fills the gaps. Ligase forms bonds between sugar-phosphate backbone. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ Replication
  • 22.
  • 23.
    Gene Expression • Synthesisof protein from DNA through RNA • The process by which DNA directs protein synthesis, gene expression includes two stages – transcription – translation
  • 24.
    Transcription and Translation •Cells are governed by a cellular chain of command – DNA → RNA → protein • Transcription – Is the synthesis of RNA under the direction of DNA – Produces messenger RNA (mRNA) • Translation – Is the actual synthesis of a polypeptide – occurs under the direction of mRNA – Occurs on ribosomes
  • 25.
    Transcription and Translation •In a eukaryotic cell the nuclear envelope separates transcription from translation • Extensive RNA processing occurs in the nucleus Eukaryotic cell. The nucleus provides a separate compartment for transcription. The original RNA transcript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. (b) TRANSCRIPTION RNA PROCESSING TRANSLATION mRNA DNA Pre-mRNA Polypeptide Ribosome Nuclear envelope
  • 26.
    Transcription • Transcription isthe DNA-directed synthesis of RNA • RNA synthesis – Is catalyzed by RNA polymerase – uracil substitutes for thymine
  • 27.
    RNA • RNA issingle stranded, not double stranded like DNA • RNA is short, only 1 gene long, where DNA is very long and contains many genes • RNA uses the sugar ribose instead of deoxyribose in DNA • RNA uses the base uracil (U) instead of thymine (T) in DNA. Table 17.1
  • 28.
    Synthesis of anRNA Transcript • The stages of transcription are – Initiation – Elongation – Termination Promoter Transcription unit RNA polymerase Start point 5′ 3′ 3′ 5′ 3′ 5′ 5′ 3′ 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ 5′ 5′ Rewound RNA R N A tr a ns cri pt 3′ 3′ Completed RNA transcript Unwound DNA RNA transcript Template strand of DNA DNA 1 Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. 2 Elongation. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5′ → 3 ′. In the wake of transcription, the DNA strands re-form a double helix. 3 Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA.
  • 29.
    • Promoters signalthe initiation of RNA synthesis • Transcription factors help eukaryotic RNA polymerase recognize promoter sequences TRANSCRIPTION RNA PROCESSING TRANSLATION DNA Pre-mRNA mRNA Ribosome Polypeptide T A T AAA A ATAT T T T TATA box Start point Template DNA strand 5′ 3′ 3′ 5′ Transcription factors 5′ 3′ 3′ 5′ Promoter 5′ 3′ 3′ 5′5′ RNA polymerase II Transcription factors RNA transcript Transcription initiation complex Eukaryotic promoters1 Several transcription factors 2 Additional transcription factors 3 Synthesis of an RNA Transcript - Initiation
  • 30.
    Synthesis of anRNA Transcript - Elongation • RNA polymerase synthesizes a single strand of RNA against the DNA template strand (anti-sense strand), adding nucleotides to the 3’ end of the RNA chain • As RNA polymerase moves along the DNA it continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides Elongation RNA polymerase Non-template strand of DNA RNA nucleotides 3′ end C A E G C A A U T A G G T T A A C G U A T C A T C C A A T T G G 3′ 5′ 5′ Newly made RNA Direction of transcription (“downstream”) Template strand of DNA
  • 31.
    • Specific sequencesin the DNA signal termination of transcription • When one of these is encountered by the polymerase, the RNA transcript is released from the DNA and the double helix can zip up again. Synthesis of an RNA Transcript - Termination
  • 32.
  • 33.
    • Most eukaryoticmRNAs aren’t ready to be translated into protein directly after being transcribed from DNA. • mRNA requires processing. • Transcription of RNA processing occur in the nucleus. – the messenger RNA moves to the cytoplasm for translation. • The cell adds a protective cap to one end, and a tail of A’s to the other end. • These both function to protect the RNA from enzymes that would degrade Post Termination RNA Processing
  • 35.
    • Most ofthe genome consists of non-coding regions called introns – Non-coding regions may have specific chromosomal functions or have regulatory purposes – Introns also allow for alternative RNA splicing • Thus, an RNA copy of a gene is converted into messenger RNA by doing 2 things: – Add protective bases to the ends – Cut out the introns Post Termination RNA Processing
  • 36.
    Alteration of mRNAEnds • Each end of a pre-mRNA molecule is modified in a particular way – The 5′ end receives a modified nucleotide cap – The 3′ end gets a poly-A tail A modified guanine nucleotide added to the 5′ end 50 to 250 adenine nucleotides added to the 3′ end Protein-coding segment Polyadenylation signal Poly-A tail3′ UTR Stop codonStart codon 5′ Cap 5′ UTR AAUAAA AAA…AAA TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide G P P P 5′ 3′
  • 37.
    RNA Processing -Splicing • The original transcript from the DNA is called pre-mRNA. • It contains transcripts of both introns and exons. • The introns are removed by a process called splicing to produce messenger RNA (mRNA)
  • 38.
    RNA Processing • Proteinsoften have a modular architecture consisting of discrete structural and functional regions called domains • In many cases different exons code for the different domains in a protein Figure 17.12 Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 1 Domain 2 Polypeptide
  • 39.
    Translation • Translation isthe RNA-directed synthesis of a polypeptide • Translation involves – mRNA – Ribosomes - Ribosomal RNA – Transfer RNA – Genetic coding - codons TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Polypeptide Amino acids tRNA with amino acid attachedRibosome tRNA Anticodon mRNA Trp Phe Gly A G C A A A C C G U G G U U U G G C Codons5′ 3′
  • 40.
    The Genetic Code •Genetic information is encoded as a sequence of nonoverlapping base triplets, or codons DNA molecule Gene 1 Gene 2 Gene 3 DNA strand (template) TRANSCRIPTION mRNA Protein TRANSLATION Amino acid A C C A A A C C G A G T U G G U U U G G C U C A Trp Phe Gly Ser Codon 3′ 5′ 3′5′
  • 41.
    The Genetic Code •Codons: 3 base code for the production of a specific amino acid, • Present on mRNA • there are 64 possible codons • 64 codons but only 20 amino acids, therefore most have more than 1 codon • 3 of the 64 codons are used as STOP signals – they are found at the end of every gene and mark the end of the protein • One codon is used as a START signal: it is at the start of every protein • Universal: in all living organisms
  • 42.
    The Genetic Code •A codon in messenger RNA is either translated into an amino acid or serves as a translational start/stop signal Second mRNA base U C A G U C A G UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Met or start Phe Leu Leu lle Val UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala UAU UAC UGU UGC Tyr Cys CAU CAC CAA CAG CGU CGC CGA CGG AAU AAC AAA AAG AGU AGC AGA AGG GAU GAC GAA GAG GGU GGC GGA GGG UGG UAA UAG Stop Stop UGA Stop Trp His Gln Asn Lys Asp Arg Ser Arg Gly U C A G U C A G U C A G U C A G FirstmRNAbase(5′end) ThirdmRNAbase(3′end) Glu
  • 43.
    Transfer RNA • Consistsof a single RNA strand that is only about 80 nucleotides long • Each carries a specific amino acid on one end and has an anticodon on the other end • A special group of enzymes pairs up the proper tRNA molecules with their corresponding amino acids. • tRNA brings the amino acids to the ribosomes Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3′ end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.) (a) 3′ C C A C G C U U A A GACACCU * G C * * G U G U *CU * G AG G U * *A * A A G U C A G A C C * C G A G A G G G * * GA CUC*AU U U A G G C G 5′ Amino acid attachment site Hydrogen bonds Anticodon A The “anticodon” is the 3 RNA bases that matches the 3 bases of the codon on the mRNA molecule
  • 44.
    Ribosomes • Ribosomes facilitatethe specific coupling of tRNA anticodons with mRNA codons during protein synthesis • The 2 ribosomal subunits are constructed of proteins and RNA molecules named ribosomal RNA or rRNA
  • 45.
    Composition of wholeribosomes and of ribosomal subunits in mammalian cells
  • 46.
    Building a Polypeptide •divide translation into three stages – Initiation – Elongation – Termination
  • 47.
    Building a Polypeptide •The AUG start codon is recognized by methionyl-tRNA or Met • Once the start codon has been identified, the ribosome incorporates amino acids into a polypeptide chain • RNA is decoded by tRNA (transfer RNA) molecules, which each transport specific amino acids to the growing chain • Translation ends when a stop codon (UAA, UAG, UGA) is reached
  • 48.
    • Translation starts(primarily) at AUG • AUG codes for methionine • 16S rRNA of the small 30S ribosomal subunit recognizes the ribosomal binding site on the mRNA -Shine-Dalgarno sequence or SD • Shine-Dalgarno sequence is complementary to sequence on 16S rRNA. • SD helps to correctly position the ribosome onto the mRNA so that the P site is directly on the AUG initiation codon INITIATION
  • 53.
    Initiation of Translation •The initiation stage of translation brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome Large ribosomal subunit The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiation factors (not shown) are required to bring all the translation components together. GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid. 2 Initiator tRNA mRNA mRNA binding site Small ribosomal subunit Translation initiation complex P site GDPGTP Start codon A small ribosomal subunit binds to a molecule of mRNA. In a prokaryotic cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, with the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine (Met). 1 Met Met U A C A U G E A 3′ 5′ 5′ 3′ 3′5′ 3′5′
  • 54.
    Amino end Growingpolypeptide Next amino acid to be added to polypeptide chain tRNA mRNA Codons 3′ 5′ Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base- pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. (c) Building a Polypeptide
  • 55.
    Elongation of thePolypeptide Chain • In the elongation stage, amino acids are added one by one to the preceding amino acid Amino end of polypeptide mRNA Ribosome ready for next aminoacyl tRNA E P A E P A E P A E P A GDP GTP GTP GDP 2 2 site site5′ 3′ TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Codon recognition. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. 1 Peptide bond formation. An rRNA molecule of the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site. 2 Translocation. The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site. 3
  • 56.
    Termination of Translation •The final stage is termination when the ribosome reaches a stop codon in the mRNA Release factor Free polypeptide Stop codon (UAG, UAA, or UGA) 5′ 3′ 3′ 5′ 3′ 5′ When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. 1 The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. 2 3 The two ribosomal subunits and the other components of the assembly dissociate.
  • 57.
    Translation • The finalstep in translation is termination. When the ribosome reaches a STOP codon, there is no corresponding transfer RNA. • Instead, a small protein called a “release factor” attaches to the stop codon. • The release factor causes the whole complex to fall apart: messenger RNA, the two ribosome subunits, the new polypeptide. • The messenger RNA can be translated many times, to
  • 58.
    A summary oftranscription and translation in a eukaryotic cell Figure 17.26 TRANSCRIPTION RNA is transcribed from a DNA template. DNA RNA polymerase RNA transcript RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. Exon Poly-A RNA transcript (pre-mRNA) Intron NUCLEUS Cap FORMATION OF INITIATION COMPLEX After leaving the nucleus, mRNA attaches to the ribosome. CYTOPLASM mRNA Poly-A Growing polypeptide Ribosomal subunits Cap Aminoacyl-tRNA synthetase Amino acid tRNA AMINO ACID ACTIVATION Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Activated amino acid TRANSLATION A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) Anticodon A C C A A A U G G U U U A U G U A CE A Ribosome 1 Poly-A 5′ 5′ 3′ Codon 2 3 4 5
  • 59.
    Post-translation • The newpolypeptide is now floating loose in the cytoplasm if translated by a free ribosme. • It might also be inserted into a membrane, if translated by a ribosome bound to the endoplasmic reticulum. • Polypeptides fold spontaneously into their active configuration, and they spontaneously join with other polypeptides to form the final proteins. • Sometimes other molecules are also attached to the polypeptides: sugars, lipids, phosphates, etc. All of these have special purposes for protein function.