Basics of Molecular Biology Central Dogma: - DNA replication - Transcription - Translation Metabolic regulation: - Genetic level - Metabolic pathway control - Cell receptor

Central Dogma Central Dogma: universal - Genetic information is stored on the DNA molecule. - This information can be replicated directly to form a second identical molecule. - Segments information on the DNA molecule can be transcribed to yield RNAs. - Using RNAs, this information is translated into proteins.

Preserving and propagating the cellular message DNA Replication Preserving and propagating the cellular message

Cell Division Cycle (Mitosis) mitotic (M) phase

Fission

DNA Replication DNA Replication : Preserve and propagate the cellular message

DNA Replication DNA helix unzips and forms two separate strands. Each strand will form a new double strands. The two resulting double strands are identical, and each of them consists of one original and one newly synthesized strand. - This is called semiconservative replication. The base sequences of the new strand are complementary to that of the parent strand.

DNA Replication DNA helix unzips and forms two separate strands. - Replication begin at the origin of replication – predetermined site. - Initiator proteins bind to the origin of replication of DNA and break hydrogen bonds of base pairs in the local region of the origin. - The two strand separate to form Y-shaped structure called a replication fork. - Movement of the fork must be facilitated by the energy-dependent action of unwinding enzymes (helicase).

DNA Replication DNA synthesis requires: - DNA template - Activated monomers: nucleoside triphosphates: dATP, dGTP, dTTP, dCTP. - an RNA primer is synthesis by primase. - DNA polymerase (Pol) Pol III: mediates the addition of nucleotides to an RNA primer. Pol I: hydrolyze an RNA primer and duplicates single-stranded regions of DNA polymer.

DNA Replication

DNA Replication DNA synthesis: - DNA polymerase works only in the 5’- to -3’ direction of the new strand: the next nucleotide is always added to the exposed 3’—OH group of the chain. - The formation of the 3’, 5’ phosphodiester bond to link the next nucleutide results in the release of a pyrophosphate proving energy for such a synthesis. - Leading strand: one strand can be formed continuously if it is synthesized in the same direction as the replication fork is moving. - Lagging strand: the other strand must be synthesized discontinuously. - DNA ligase joins the two short pieces of DNA on the continuous strand.

3’ 5’ 3’ DNA chain

http://www.ncc.gmu.edu/dna/repanim.htm

Transcription: sending the message The primary product of transcription: m-RNA, t-RNA and r-RNA. RNA polymerases consist of : sigma factor: a protein locate the beginning for the message. core enzyme: it contains the active sites. Read 3’ to 5’ direction of DNA template RNA is synthesized in the direction of 5’ to 3’. Both strands of DNA could be transcribed. The base-sequence of RNA is the precise complement of the DNA template sequence. DNA template RNA product A U T A G C C G

Transcription Transcription requires: RNA polymerases Growth of RNA polymers is energy requiring. Activated ribonucleotide triphosphate: ATP, GTP, UTP, CTP.

Transcription Process Initiation: The sigma factor recognizes a specific sequence of nucleotides on a DNA strand – promoter region. the strands unwound. Elongation: Transcription starts with the core enzyme then the sigma factor is released. Termination: RNA polymerase encounter a stop signal or transcription terminator (in some case rho protein is required for termination). - the RNA polymerase dissociate from the DNA template - the RNA transcript is released.

http://www.ncc.gmu.edu/dna/mRNAanim.htm

Difference in Transcription Between Procaryotes and Eucaryotes Related protein are encoded in a row without interspacing terminators. Transcription from a single promoter may result in a polygenic message containing many genes. Regulation from a single promoter provide a efficient regulation of functional related protein. - No physical separation of chromosome and ribosome : m-RNA bind to ribosome and begin translation while the transcription is still on going.

http://micro.magnet.fsu.edu/cells/procaryotes/images/procaryote.jpg

Difference in Transcription Between Procaryotes and Eucaryotes The DNA can encode for a transcript with an intervening sequence called intron in the middle of the transcription. - intron cuts out mRNA at two specific sites - after it degraded, the spliced RNA fragments could be joined by a process called m-RNA splicing. - The spliced message can then be translated into an actual protein. - Once mRNA is recovered from the cytosol, it is mature while it within the nucleus has introns. - introns likely play a role in either evolution or cellular regulation.

Difference in Transcription Between Procaryotes and Eucaryotes Eucaryotes: (continued) Two modification of mRNA: RNA capping: the 5’ end is modified by the addition of a guanine nucleotide with a methyl group attached. Polyadenylation: a string of adenine nucleotides are added to the 3’ end. The string is several hundred nucleotides long. These two modifications are thought to increase mRNA stability and facilitate transport across the nuclear membrane.

Eucaryote cell structure

Translation Translation is the final step on the way from DNA to protein. It is the synthesis of proteins directed by a mRNA template. The information contained in the nucleotide sequence of the mRNA is read as three letter words (triplets), called codons. Each word stands for one amino acid. During translation amino acids are linked together to form a polypeptide chain which will later be folded into a protein.

Translation: Message to Product Universal three-letter codons on mRNA: A, G, C, U - 64 codes for 20 standard amino acids - more than one codon can specify a particular amino acids. - Nonsense codons: UAA, UAG and UGA - Do not encode normally for amino acids. - Act as stop points in translation. - encoded at the end of each gene.

Translation

http://www.phschool.com/science/biology_place/biocoach/translation/init.html

Translation Important Components: Ribosome: The ribosome is the cellular factory responsible for the protein synthesis. It consists of two different subunits, one small and one large and is built up from rRNA and proteins. sedimentation coefficients – rough mass determination: 30S (small subunits) and 50S (big subunits) for procaryotes 40S (small subunits) and 60S (big subunits) for eucaryotes Inside the ribosome the amino acids are linked together into a chain through multiple biochemical reactions.

Translation-components t-RNA: The charged t-RNA (aminoacyl-t-RNA) carries an amino acid at one end and has a triplet of nucleotides, an anticodon, at the other end. It is formed by the energy from two phosphate bonds and enzymes (aminoacyl-t-RNA synthetases) The anticodon of a t-RNA molecule can basepair, i.e form chemical bonds, with the m-RNA's three letter codon. The t-RNA acts as the translator between m-RNA and protein by bringing the specific amino acid coded for by the m-RNA codon.

t-RNA

Translation-components m-RNA template The translation process also involves a large number of protein factors that facilitate binding of mRNA and tRNA to the ribosome. Protein synthesis consumes a large part of the energy produced in the cell.

Translation Processes Translation consists of Initiation, Elongation and Termination. Initiation results in the formation of an initiation complex in which the ribosome is bound to the specific initiation (start) site on the mRNA while the initiator tRNA charged with (N-formyl)methionine is annealed to the initiator codon and bound to the ribosome. - Protein synthesis begins with a AUG codon (less frequently GUG) on the m-RNA AUG encodes for a modified methionine, N-formylmethionine (fMet). In the middle of protein, AUG encodes for methionine. http://www.phschool.com/science/biology_place/biocoach/translation/init.html

Translation Processes To recognize the initiation AUG: Procaryotes: the AUG is preceded about ten nucleotides away by a purine-rich RNA sequence that base-pairs with a complementary sequence in a ribosomal RNA molecule. Eucaryotes: the AUG closest to the 5’ end of an mRNA. m-RNA Purine-rich (-10) AUG (+1) 5’ Protein fMet AUG (+1) cap 5’ m-RNA fMet Protein A procaryotic mRNA molecule can have a number of start sites while a eucryotic mRNA has only one start site.

http://www.phschool.com/science/biology_place/biocoach/translation/init.html http://www.phschool.com/science/biology_place/biocoach/translation/elong1.html

Translation Processes Elongation joins amino acids to the growing polypeptide chain according to the sequence specified by the message. The formation of the peptide bond between the two amino acids occurs on adjacent sites on the ribosome: the P or peptidyl site and the A or aminoacyl site. The growing protein occupies the P site, while the next amino acid to be added occupies the A site. As the peptide bond is formed, the t-RNA associated with the P site is released.

Translation Processes Elongation (continued) As the peptide bond is formed, the t-RNA associated with the P site is released. m-RNA was moved down one codon so as to cause the t-RNA in the A site to be in the P site. The next charged t-RNA with the correct anticodon can be recognized and inserted into the A site. The whole process is repeated until a nonsense code or stop codon is reached. The cell requires four phosphate bonds to add one amino acid to each growing polypeptide: two to charge the t-RNA and two in the process of elongation

http://www.phschool.com/science/biology_place/biocoach/translation/elong1.html

Translation Processes Termination At a stop codon, a release factor reads the triplet, and polypeptide synthesis ends. the polypeptide is released from the tRNA. the tRNA is released from the ribosome, the two ribosomal subunits separate from the mRNA.

http://www.phschool.com/science/biology_place/biocoach/translation/term.html

More than one ribosome can translate an mRNA at one time, Several ribosomes can translate an procaryotic mRNA at the same time, forming what is called a polysome. More than one ribosome can translate an mRNA at one time, making it possible to produce many polypeptides simultaneously from a single mRNA.

Posttranslation Posttranslational modification means the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis. It may involve the folding of a proper structure, the formation of disulfide bridges and attachment of any of a number of biochemical functional groups, such as phosphate, acetate, various lipids and carbohydrates. e.g. phosphorylation for controlling the behavior of a protein, for instance, activating or inactivating an enzyme. (TP53 function as tumor suppressor).

Posttranslation In procaryotes, posttranslation can take place when proteins are secreted through a membrane (cytoplasimc membrane or outer membrane) (cotranslationally) Such proteins exist in a pre-form from translation which is the signal sequence plus the mature form of protein. A signal sequence is about 20-25 amino acids and is clipped off during secretion.

Posttranslation In eucaryotes, proteins are released by exocytosis – a process for a cell to get rid of the large molecules through its membrane. Transport vesicles - carry proteins and other chemicals from endoplasmic reticulum bound with ribosomes to golgi apparatus and other membrane-bound compartment. Posttranslation take places in these organelles. - fuse with the plasma membrane - release their contents

Posttranslation - Only protein with a signal sequence can enter secretory pathway. - Two pathways: - constitutive exocytosis: operates at all time. - regulated exocytosis: operates in specialized secretory cells.

Posttranslation Some posttranslation can only occurs in eucaryotes. e.g.N-linked glycosylation involves in endoplasmic reticulum and golgi apparatus. It can serve to target the protein to a particular compartment or to control its degradation and removal from the organism.

Summary of Central Dogma DNA replication DNA-RNA transcription RNA-Protein translation Posttranslation components, processes, features

Summary DNA replication RNA transcription Translation Initiation Elongation (synthesis) Termination Components (monomers and other components)