DNA Replication PDF

 DNA REPLICATION DNA REPLICATION IN PROKARYOTES DNA replication in E. coli and other prokaryotes occurs by a semiconservative mechanism in which the strands of a DNA double helix separate and a new complimentary strand of DNA is synthesized on each of the two parental template s...

 DNA REPLICATION DNA REPLICATION IN PROKARYOTES DNA replication in E. coli and other prokaryotes occurs by a semiconservative mechanism in which the strands of a DNA double helix separate and a new complimentary strand of DNA is synthesized on each of the two parental template strands. Semiconservative replication results in two double-stranded DNA molecules, each of which has one strand from the parent molecule and one newly synthesized strand. DNA synthesis enzymes Four components are needed for the in vitro synthesis of DNA. If anyone is missing, DNA synthesis would not occur: 1. All four deoxyribonucleoside 5’ triphosphates (dATP, dGTP, dTTP, dCTP) dNTPs. If anyone is missing, synthesis will not occur. 2. Magnesium ions (Mg2+) MgCl2 as a cofactor 3. A fragment of DNA to act as a template 4. DNA polymerase Molecular details of DNA replication The initiation of DNA replication in prokaryotes requires the local denaturation of the DNA at a specific DNA sequence called an origin of replication. At that site the double helix denatures into single strands, exposing the bases for the synthesis of new strands. In a circular chromosome, such as is found in E. coli, the local denaturing of the DNA produces what is called a replication bubble. In E. coli there is a single origin, or replication called oriC from which replication proceeds bidirectionally. An initiator protein binds to the parental DNA molecule at the origin of replication sequence. The two DNA strands then separate in that region; this involves a localized Denaturation and requires the physical untwisting of the DNA. This untwisting (uses energy derived from the hydrolysis of ATP) is catalyzed by the enzyme DNA helicase. Another enzyme, DNA primase binds to the helicase and the denatured DNA. Primase is derived from RNA polymerase. The initiation of DNA synthesis involves the synthesis of a short RNA primer, catalyzed by primase. The complex of the primase, helicase, and perhaps other proteins with the DNA is called the primosome. The primer nucleotide serves as a substrate for the action of DNA polymerase. Short DNA segments called Okazaki fragments remove the RNA primers. The new DNA is made in the 5’ to 3’ direction. DNA REPLICATION IN EUKARYOTES It’s similar as in prokayotes. However the added complication is that DNA is distributed among many chromosomes in eukaryotes. In each cell division cycle, each of these chromosomes must be faithfully duplicated and a copy of each distributed to each of the two progeny cells. Recall the cell cycle: G1; S; G2; M. The DNA replicates during the S phase, and the progeny chromosomes segregate into daughter cells during the M phase. Molecular details of DNA synthesis in Eukaryotes Nucleosome. Replication of the eukaryotic chromosome must involve the replication of the DNA and of the histone core of the nucleosome as well as a doubling of the non-histones. The sequential steps described for the DNA synthesis in prokaryotes also occur for DNA synthesis in Eukaryotes, namely, Denaturation of the DNA double helix and the Semiconservative, semidiscontinous replication of the DNA. Initiation of DNA replication If there was only one origin of replication per chromosome, the replication of each chromosome would take many, many hours. Gestation would be many years instead of 9 months. DNA replication is initiated at many origins of replication throughout the genome. At each origin of replication, the DNA denatures. Replication proceeds bidirectionally, and the DNA double helix opens to expose single strands that act as templates for new DNA synthesis. Eventually each replication fork will run into an adjacent replication fork. Because A-T base pairs have two H bonds, it’s easier to disrupt than G-C base pairs with three H bonds. Single-strand binding (SSB) protein prevents the DNA from renaturing. These SSB proteins stimulate polymerase activity.  TRANSCRIPTION Transcription is the transfer of information from a double- stranded, template DNA molecule to a single-stranded RNA molecule. TRANSLATION Translation (protein synthesis) is the conversion, in the cell, of the messenger RNA (mRNA) base sequence information into the amino acid sequence of a polypeptide. All base pairs in the genome are replicated during the DNA synthesis phase of the cell cycle, but only some of the base pairs are transcribed into RNA. The specific sequence of base pairs that are transcribed are called genes, and thus the transcription process is also referred to as gene expression. Around the beginning and end of each gene are base-pair sequences called gene regulatory elements, which are involved in the regulation of gene expression. TRANSCRIPTION Four major classes of RNA molecules or transcripts are produced by transcription: 1. messenger RNA (mRNA); 2. transfer RNA (tRNA); 3. ribosomal RNA (rRNA); 4. small nuclear RNA (snRNA). The first three are found in both prokaryotes and eukaryotes, while snRNA are only found in eukaryotes. Only the mRNA molecule is translated to produce a protein molecule. A gene that codes for an mRNA molecule, and hence for a protein, is called a structural gene or a protein- coding gene. Transcription is catalyzed by an enzyme called RNA polymerase. The DNA double helix must unwind before transcription can begin. Only one of the two DNA strands is transcribed into an RNA. This strand is called the template strand and the other is the non-template strand or coding strand. In transcription, the RNA precursors are the ribonucleoside triphosphates ATP, GTP, CTP, and UTP collectively called NTPs. RNA polymerization is very similar to the DNA polymerization reaction. The next nucleotide to be added to the chain is selected by the RNA polymerase for its ability to pair with the exposed base on the DNA template strand. As in DNA replication, RNA is synthesized in the 5’ to 3’ direction. To initiate transcription of a gene, RNA polymerase binds to a transcription-controlling sequence adjacent to the start of the gene; this sequence is called the promoter. The sigma factor is essential for promoter recognition. If sigma is absent, the core enzyme initiates transcription randomly. Transcription ceases when a controlling element called a transcription terminator sequence or a terminator is encountered at the end of a gene. An RNA polymerase enzyme performs several functions:  It recognizes a promoter on the double-stranded DNA.  It causes the DNA to denature and unwind into single strands at the promoter (similar to the initiation of DNA replication).  As a result of reading the promoter sequence, the RNA polymerase orients itself properly and transcribes the entire template strand of the gene.  Lastly it stops transcribing when it reaches and recognizes the terminator.  Promoter – RNA-coding sequence – Terminator Transcription in prokaryotes and eukaryotes are similar. The main differences are that more than one RNA polymerase enzyme occurs in eukaryotes and that the transcription sequences (promoters and terminators) are different. Initiation – Elongation – Termination In prokaryotes, the RNA copy of a gene is messenger RNA, ready to be translated into protein. In fact, translation starts even before transcription is finished. In eukaryotes, the primary RNA transcript of a gene needs further processing before it can be translated. This step is called “RNA processing” or “post transcriptional modifications”. Also, it needs to be transported out of the nucleus into the cytoplasm. rRNA links amino acids together to form proteins. The cap is 7- methylguanosine which helps to protect from attack by ribonucleases. Splicing is catalysed by large protein complexes called spliceosome, assembled from proteins and snRNA that recognize splice sites. Poly A tail protects the 3’ end from ribonuclease digestion.  TRANSLATION THE GENETIC CODE The conversion in the cell of the mRNA base sequence information into an amino acid sequence of a polypeptide is called translation. The DNA base-pair information that specifies the amino acid sequence of a polypeptide is called the genetic code. The nature of the genetic code mRNA molecule are read in groups of three called codons to specify the amino acid sequence in protein. Four different nucleotides (A,C,G,U) occur in the message and 20 different amino acid occur in protein. If it were a one-letter code then, only four amino acids could be coded. If it were a two-letter code, then it would be 16 amino acids. A three-letter code generates 64 possible codes. Some amino acids may be specified by more than one codon. The characteristics of the genetic code are as follows: 1. The code is a triplet code. 2. The code is comma-free. The mRNA is read continuously, three nucleotides (one codon) at a time, without skipping any nucleotide of the message. 3. The code is nonoverlapping. AAGAAGAAG lysine 4. The code is almost universal. All organisms share the same genetic language. 5. The code is degenerate. With two exceptions AUG methionine and UGG tryptophan, more than one codon occurs for each amino acid. This multiple coding is called the degeneracy of the code. 6. The code has start and stop signals. Methionine is most commonly used as a start codon for protein synthesis. 61 of the 64 codons specify amino acids. These codons are called the sense codons. The other three codons – UAG (amber); UAA (ochre); and UGA (opal). These are called stop codons or nonsense codons or chain-terminating codons. TEST #2