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 Represents information about a reaction that is independent of enzymes that catalyze the reaction
 Connected to enzyme(s) via enzymatic reaction frames
 Classified with EC system when possible
 Example: 2.7.7.7 – DNA-directed DNA polymerization
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Carried out by five enzymes in E. coli
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 A necessary bridge between enzymes and
“generic” versions of reactions
 Carries information specific to an enzyme/reaction combination:

Cofactors and prosthetic groups
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Alternative substrates
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Links to regulatory interactions
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Kinetic data (Km, Vmax, etc.)
 Frame is generated when protein is associated with reaction (via protein or reaction editor)
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 Left/Right reflect direction of reaction as written by Enzyme Commission
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Reflects systematic direction for different reaction classes
 Left/Right do not necessarily correspond to physiological direction of a reaction
 Get-rxn-direction(rxn)
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Returns :L2R or :R2L or :BOTH or NIL
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Integrates all available info about direction of this reaction
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
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Direction explicitly curated for reaction
Direction(s) it occurs in all pathways in the PGDB
Direction(s) as specified in Enzymatic-Reactions
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 Balance-state
 EC-number
 Enzymatic-reaction
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Generated in protein or reaction editor
 In-pathway
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Generated in pathway editor
 Left and Right
 Reaction-Direction
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:left-to-right, :right-to-left, :reversible
 Spontaneous?
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 Enzyme
 Reaction
 Regulated-By
 Cofactors
 Kinetic slots:
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Vmax, Km, Kcat, Specific-Activity, Temperature-Opt, pH-Opt
 Reaction-Direction
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Reaction may be reversible, but this enzyme effectively only catalyzes it in one direction.
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 Genes-of-reaction (rxn)
 Substrates-of-reaction (rxn)
 Enzymes-of-reaction (rxn)
 Lacking-ec-number (organism)
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Returns list of rxns with no ec numbers in that database
 Get-reaction-direction-in-pathway (pwy rxn)
 Reaction-type(rxn)
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Indicates types of Rxn as: Small molecule rxn, transport rxn, protein-small-molecule rxn
(one substrate is protein and one is a small molecule), protein rxn (all substrates are proteins), etc.
 All-rxns(type)
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Specify the type of reaction (see above for type)
 Obtain-rxn-stats
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Returns six values
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Length of : all-rxns, transport, non-transport, etc…
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 Find all small-molecule reactions that have no enzyme but are not spontaneous (“orphan” reactions)
 Find all reactions that consume a given compound
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 Find all small-molecule reactions that have no enzyme but are not spontaneous (“orphan” reactions):
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(loop for r in (all-rxns :small-molecule) when (and (not (slot-has-value-p r 'enzymatic-reaction)
(not (get-slot-value r 'spontaneous?))) collect r)
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 Find all reactions that consume a given compound:
(defun rxns-consuming-cpd (cpd)
(append
(loop for r in (get-slot-values cpd ‘appears-in-left-side-of) for dir = (get-rxn-direction r) if (or (eq dir :l2r) (eq dir :both)) collect r)
(loop for r in (get-slot-values cpd ‘appears-in-right-side-of) for dir = (get-rxn-direction r) if (or (eq dir :r2l) (eq dir :both)) collect r)))
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
PGDBs only represent RNAs that are “terminal gene products”
 tRNAs
 rRNAs
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Regulatory RNAs
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Miscellaneous small RNAs
 Slots similar to proteins
 tRNAs can have an anticodon
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 An ordered set of interconnected, directed biochemical reactions
 Reactions form a coherent unit, e.g.
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Regulated as a single unit
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Evolutionarily conserved across organisms as a single unit
When combined, perform a single cellular function
Historically grouped together as a unit
 Includes metabolic pathways and signaling pathways
 Evidence for all reactions in a single organism
 Pathways can be linear, cyclical, branched, or some combination
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 REACTION-LIST: unordered list of reactions that comprise the pathway
 PREDECESSORS: list of reaction pairs that define ordering relationships between reactions.
E.g. R1 R2 C
A B
R3 D
(R2 R1) : Predecessor of R2 is R1
(R3 R1) : Predecessor of R3 is R1
(R1) : R1 has no predecessor (can be omitted)
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 Reaction directions
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Some reactions are unidirectional, but many are reversible – how do we know in which direction to draw the reaction?
 Main vs. side substrates
A B C
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D E F
Main compounds form the backbone of the pathway

 substrates shared between connecting reactions major inputs and outputs.
Side compounds omitted from pathway diagrams at low detail levels
Individual reactions do not necessarily have main and side compounds – a particular substrate may be either a main or a side depending on the pathway context.
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Our philosophy: Enable curator to specify as little as possible. Compute as much as possible. This reduces redundancy and potential for inconsistencies.
Example:
Reactions R1: A + B  C + D
R2: B  E
Predecessors: (R2 R1)
 Only substrate overlap is B
 B must be a main substrate
 A must be a side substrate,
 R1 must proceed from right to left
 R2 must proceed from left to right
C + D  B  E
A
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Unfortunately, mains, sides and reaction directions are sometimes ambiguous:
 At beginnings and ends of pathways
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Use heuristics to determine main/side substrates at beginnings, ends of pathways
Not always what the curator wants
 Substrate overlap with both sides of a reaction, e.g. A + B  C + D
C + B  E
 Solution: Additional slot PRIMARIES, should only be populated when necessary:
PRIMARIES: (R (A B) (C)) says that for reaction R, A and B are both main reactants, and C is a main product.
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 ENZYMES-NOT-USED: a reaction may be catalyzed by multiple enzymes, but not all the enzymes necessarily participate in a given pathway
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Not present in the same compartment with rest of pathway enzymes
Down-regulated or not expressed under conditions in which pathway is active
ENZYMES-NOT-USED slot lists enzymes that are not involved in the pathway even though they catalyze one of its reactions.
 LAYOUT-ADVICE: helps software draw pathway correctly, e.g. in a cyclical pathway, tells which substrate should be at the top.
 HYPOTHETICAL-REACTIONS: list of reactions in the pathway that are considered hypothetical (i.e. no direct experimental evidence)
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…  X [n] X [n+1] X [10]
 POLYMERIZATION-LINKS: specifies reactions that should be connected by a polymerization link
(X R1 R1) --- REACTANT-NAME-SLOT: N-NAME
--- PRODUCT-NAME-SLOT: N+1-NAME
 CLASS-INSTANCE-LINKS: specifies when a link should be drawn between a substrate class and some instance of it (necessary only if instance is not a member of some reaction, so no predecessor relationship can be defined)
R1 --- PRODUCT-INSTANCES: X [10]
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 Can be used as an alternative or in addition to defining super-pathways
 Link must be to or from some main substrate in the pathway
 Other end of link can be a pathway, a reaction, or an arbitrary text string
 Software automatically computes direction of link, but curator can override it
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 Collection of pathways that connect to each other via common substrates or reactions, or as part of some larger logical unit
 Can contain both sub-pathways and additional connecting reactions
 Can be nested arbitrarily
 REACTION-LIST: a pathway ID instead of a reaction ID in this slot means include all reactions from the specified pathway
 PREDECESSORS: a pathway ID instead of a tuple in this slot means include all predecessor tuples from the specified pathway
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 Signaling pathways have different layout conventions than metabolic pathways
 Layout is done manually by curator using specialized editor
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Curator has more control
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Lack of automatic layout algorithm means that diagram won’t update automatically when data changes.
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See http://bioinformatics.ai.sri.com/ptools/ptools-resources.html
(all-pathways)
(base-pathways)
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Returns list of all pathways that are not super-pathways
(genes-of-pathway pwy)
(unique-genes-of-pathway pwy)
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Returns list of all genes of a pathway that are not also part of other pathways
(enzymes-of-pathway pwy)
(compounds-of-pathway pwy)
(get-reaction-list pwy)
(variants-of-pathway pwy)

Returns all pathways in the same variant class as a pathway
(get-predecessors rxn pwy), (get-successors rxn pwy)
(get-rxn-direction-in-pathway pwy rxn)
(pathway-inputs pwy), (pathway-outputs pwy)
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Returns all compounds consumed (produced) but not produced (consumed) by pathway (ignores stoichiometry)
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 Find all genes involved in metabolic pathways
 Find all compounds that are unique to a single pathway
 Find the reactions of a pathway that have multiple isozymes
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 Find all genes involved in metabolic pathways:
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(remove-duplicates
(loop for p in (all-pathways) append (genes-of-pathway p)))
 Find all compounds that are unique to a single pathway:
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(loop for p in (base-pathways) append (loop for c in (compounds-of-pathway p) when (null (remove p (pathways-of-compound c))) collect (list c p)))
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 Find the reactions of a pathway that have multiple isozymes:
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(defun rxns-w-multiple-isozymes (pwy)
(loop for rxn in (get-reaction-list pwy) for enzymes = (enzymes-of-reaction rxn) when (> (length enzymes) 1) collect rxn))
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 Class Regulation with subclasses that describe different biochemical mechanisms of regulation
 Slots:
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Regulator
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Regulated-Entity
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Mode
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Mechanism
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 Class Regulation-of-Enzyme-Activity
 Each instance of the class describes one regulatory interaction
 Slots:
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Regulator -- usually a small molecule
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Regulated-Entity -- an Enzymatic-Reaction
Mechanism -- One of:

Competitive, Uncompetitive, Noncompetitive, Irreversible, Allosteric,
Unkmech, Other
Mode -- One of: + , -
Physiologically-Relevant?
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Transcription-Unit
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One or more genes, transcribed as a unit
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Any gene, promoter, etc. can belong to multiple transcription-units
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One or zero promoters
Zero or more terminators, binding-sites
Transcription-Direction: +, -
Promoter
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Absolute-plus-1-pos = transcription start site
Binds-Sigma-Factor
Terminator
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Rho-Dependent or Rho-Independent
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Left-End-Position, Right-End-Position
Binding-Site
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DNA-Binding-Site or mRNA-Binding-Site
Left-End-Position, Right-End-Position
Involved-in-Regulation
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 Class Transcription-Factor-Binding
 Slots:
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Regulator -- instance of Proteins or Complexes (a transcription-factor)
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Regulated-Entity -- instance of Promoters or Transcription-
Units or Genes

Mode -- One of: + , -

Associated-Binding-Site
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 Class Transcriptional-Attenuation
 Several subclasses depending on type of attenuation
 Slots common to all:
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Regulator -- Depends on subtype of attenuation
Regulated-Entity -- instance of Terminators or Genes or
Transcription-Units
Mode -- One of: + , -
 Slots particular to one or more subclasses:


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Associated-Binding-Site
Antiterminator-Start-Pos, Antiterminator-End-Pos
Pause-Start-Pos, Pause-End-Pos
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Protein-Mediated-Attenuation
RNA-Mediated-Attenuation
Small-Molecule-Mediated-Attenuation
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Regulator = A protein/RNA/small molecule
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Leader transcript binds protein/RNA/small molecule and determines formation of terminator or antiterminator
Ribosome-Mediated-Attenuation
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Regulator = charged tRNA
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Ribosome pauses, determining whether terminator or antiterminator forms
RNA-Polymerase-Modification
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Regulator = instance of Proteins or Complexes
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Regulatory protein binds to site in transcription unit and interacts with RNA polymerase to determine termination
Rho-Blocking-Antitermination
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 Class Regulation-of-Translation
 Several subclasses depending on type of attenuation
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Compound-Mediated (i.e. riboswitch)
RNA-Mediated
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Protein-Mediated
 Slots:
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Regulator -- Depends on subtype of attenuation
Regulated-Entity -- Transcription-Unit or Gene
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Mode -- One of: + , -
Mechanism – Translation-Blocking, mRNA-Degradation, and/or
Translation-Attenuation
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Associated-Binding-Site
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 (transcription-unit-genes tu)
 (transcription-unit-promoter tu)
 (transcription-unit-binding-sites tu)
 (transcription-unit-mrna-binding-sites tu)
 (transcription-unit-terminators)
 (transcription-unit-transcription-factors)
 (terminators-affecting-gene gene)
 (containing-tus frame)
 (binding-site-transcription-factors bs)
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 (genes-regulating-gene gene)
 (genes-regulated-by-gene gene)
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Includes all transcriptional or translational regulation
 (direct-activators frame)
 (direct-inhibitors frame)
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Frame will be an enzymatic-reaction, transcription-unit, promoter, etc. – this is a low-level function
 (transcription-unit-activators tu)
 (transcription-unit-inhibitors tu)
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Includes transcriptional and translational regulators, and regulators of promoter as well as direct regulators of tu
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 Find all DNA-binding-sites that regulate a gene
 Find only those substrate-level regulators of an enzyme that are considered physiologically relevant
 Find all inhibitors of an enzyme, including transcriptional, translational and substrate-level inhibition.
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 Find all DNA-binding-sites that regulate a gene:
(defun gene-binding-sites (gene)
(remove-duplicates
(loop for tu in (containing-tus gene) append (transcription-unit-binding-sites tu))))
 Find only those substrate-level regulators of an enzyme that are considered physiologically relevant:
(defun relevant-regulators (enz)
(loop for enzrxn in (get-slot-values enz ‘catalyzes) append (loop for regframe in (get-slot-values enzrxn ‘regulated-by) when (get-slot-value regframe ‘physiologically-relevant?) collect (get-slot-value regframe ‘regulator)
)))
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 Find all inhibitors of an enzyme, including transcriptional, translational and substrate-level inhibition:
(defun enzyme-inhibitors (enz)
(let* ((genes (genes-of-enzyme enz))
(tus (remove-duplicates
(loop for g in genes append (containing-tus g))))
(terminators
(remove-duplicates
(loop for g in genes append (terminators-affecting-gene g))))
(enzrxns (get-slot-values enz 'catalyzes))
)
(append (loop for enzrxn in enzrxns append (direct-inhibitors enzrxn))
(loop for tu in tus append (transcription-unit-inhibitors tu))
(loop for term in terminators append (direct-inhibitors term))
)))
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