Reproduction

Cell cycle Transcription Translation Metabolic Regulation - Cellular Level Metabolic Regulation - Genetic Level Genetic Level Metabolic Pathway Control and MCA

Objectives
understand the basic differences in the reproduction of prokaryotes and eukaryotes grasp the central dogma of reproduction understand the role of DNA and RNA in reproduction and discuss the two main steps in protein synthesis recognise the need for and the main types of posttranslational protein processing distinguish two levels of the metabolic regulation understand the importance of MCA in the optimisation of protein production

Environment

s gar Su

Proteins

CATABOLISM

AAcids

Precursors NAcids Carriers

FAcids
Cofa ctors

NUTRIENTS

Trans* POLYMERISATION

Genes

SIGNAL
R PTO E EC R

BIOLOGICAL EFFECT

PROTEIN Translation mRNA Processes occur over different timescales

Signal Transduction

Transcription DNA

Molecular reproduction
A number of events has to take place before the cell can divide, as the daughter cell has to have all the necessary constituents (organelles) Most importantly, the genetic information which controls the behaviour of the cell, has to be copied Genetic information is contained in DNA in a number of distinct structural elements - chromosomes. Each eukaryotic cells contains a number of pairs of chromosomes specific for the species: human - 23 pairs (=46 chromosomes) which measure 230 mm stretched and 5 m packed.

Number of chromosomes/organism
Ant Barley Hare Chicken Carp 2 14 46 78 104 Droshophila Maize Gorilla Dog 8 20 48 78

Ichthyomys pittieri 94 (Highest mammal) (semiaquatic rodent) Ophioglossum 1200 (Highest plant) reticulatum (fern) Aulacantha (protozoa) 1600 (Highest overall)

Genome complexity

Genetic code
As it is necessary to code 20 different AAs and DNA has only 4 different nucleotides, a three letter code is required 2-letter code would only give 42 = 16 AAs Codon is the 3-letter combination coding the genetic information in DNA, i.e. 43 = 64 combination are available, more than required. 61 of these are used to code the AAs (some are coded for by a number of codons) and 3 are non-sense codons used to control the transcription. first of all DNA has to be replicated (copied exactly) to transfer genetic information to the daughter cell this process is semiconservative, i.e. old strand serves as a template for the synthesis of the new strand

Protein synthesis
Central dogma of reproduction
The flow of info from DNA into protein structure has to be intermediated DNA is either contained in the nucleus (eukaryotes) or attached to the membrane (prokaryotes) and its integrity needs to be protected.

Transcription

Translation

DNA

mRNA

protein

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Transcription
The product of transcription is m-RNA mediated by RNA-polymerase.

Translation
It progresses in the following stages: 1) Initiation - m-RNA binding to ribosomes. Protein synthesis begins with AUG codon (coding for N-formylmethionine). The cell knows whether it is and initiator AUG or proper AUG (coding for Met) according to ribosome binding site - charged t-RNAs (charged with AA) use their decoder (anticodon) end which has a nucleotide sequence complimentary to the m-RNA codon to bind onto the m-RNA in a particular order. There is a P (peptidyl) site and A (aminoacyl) site on the ribosome. t-RNA binds onto A site and using energy from 2 ~P bonds, AA binds to the existing peptide. t-RNA which was attached to the P site and held the existing polypeptide is released and t-RNA that just delivered its AA moves to P site.

2) Elongation

3) Termination -when a nonsense (stop) codon is detected, the release factor RF releases the protein from the ribosome.

translation

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Post-translational processing
Often the polypeptide formed from the ribosome must undergo further processing to become active (must be folded into the proper structure, several chains may have to be associated together, etc.). The protein is often translocated across a membrane either cotranslationally (during translation) or posttranslationally. To cross a membrane, proteins usually have a signal sequence (20-25 AAs) which is clipped off during secretion to produce the mature protein. Sometimes the protein have to be in a particular form (phosphorylated (having P), glycosylated (having and associated sugar), etc.). These modifications can be quite complex and often influence the choice of host organisms for the production of proteins.

Proteins for secretion (red dots) are synthesised by rER ribosomes (blue dots) Pro injected into the lumen of ER or into its membrane encapsulated in vesicles, fuse with cis Golgi network after further processing, the trans Golgi network sorts them to the secretory vesicles or lysosomes

Regulation Genetic Level

The most common control on genetic level is the transcriptional control (i.e. increased or decreased rate of transcription of the relevant gene). Some enzymes necessary for basic cell functions (e.g. glycolysis) are produced constitutively (unregulated genes), but most are regulated by: a) feedback repression - the end product of the reaction accumulates and blocks transcription b) induction - a metabolite (often the S) accumulates and acts as an inducer for transcription. In both cases a repressor protein together with corepressor (usually end product of a pathway) blocks the operator region of mRNA and stops transcription. Inducer binds to repressor and releases it from mRNA enabling transcription. Several genes can be under the control of one promoter. A set of contiguous genes, encoding proteins with related functions under the control of a single promoter-operator is called operon, e.g. lac operon controls the synthesis of 3 proteins involved in lactose utilisation as a C source in E. coli.

Metabolic Regulation
Irrespective whether an enzyme is made from a regulated or constitutive gene, its activity in the cell is still regulated at the metabolic pathway regulation While cell is trying to make the most effective use of its resources, the fermentation scientists are trying to disrupt this control strategy and overproduce a product of interest. It is therefore essential to understand the control of metabolic pathways in order to optimise the industrial bioprocess development MCA (studying fluxes through pathways) Essentially, the cell controls the activity of enzymes through feedback control

Metabolic Pathway Control

1) Feedback inhibition

E S
2) Isoenzymes

E1 S E2 M1

E3 M2

E4

P1 P2

E5

Metabolic Pathway Control

1) Concentrated Feedback

E1 S
2) Sequential Feedback

E3 M1 M2

E4

P1 P2

E5

E1 S M1

E3 M2

E4

P1 P2

E5