Question

Compare and contrast mechanisms of transcriptional repression in prokaryotes and eukaryotes. Be sure to mention the involvement of cis-acting and trans-acting factors.

Answer 1

THE GENE IS FIRST TRANSCRIBED INTO MRNA, THEN TRANSLATED INTO A POLYPEPTIDE CHAIN. THAT POLYPEPTIDE IS A COMPONENT OF THE PROTEINS THAT MAKE UP YOUR BODY, INCLUDING YOUR HEIGHT, FACIAL FEATURES, AND EVERYTHING ELSE. WHEN THE CODES HIDDEN INSIDE OUR GENES COME OUT TO LIGHT AS PHYSICAL TRAITS, WE CALL IT GENE EXPRESSION.

WHEN GENES EXPRESS THEMSELVES, THEY PUT OUT PROTEIN MOLECULES THAT RESULT FROM THE GENETIC CODES THEY HAVE INSIDE. GENES EXPRESS THEMSELVES BY TURNING THE DNA CODE INTO A PROTEIN BY WAY OF TRANSCRIPTION AND TRANSLATION. IT'S BASICALLY ANOTHER WAY OF TALKING ABOUT THE CENTRAL DOGMA.

PROKARYOTIC VS. EUKARYOTIC TRANSCRIPTION

GENE REGULATION HAPPENS DIFFERENTLY DEPENDING ON WHETHER THE ORGANISM IS A PROKARYOTE OR A EUKARYOTE. EUKARYOTES ARE ORGANISMS, LIKE PLANTS, ANIMALS, FUNGI AND PROTISTS, THAT ALL HAVE CELLS WITH NUCLEI AND ORGANELLES INSIDE. MOST EUKARYOTES ARE MULTICELLULAR. A PROKARYOTE IS A SINGLE-CELLED ORGANISM, LIKE BACTERIA, THAT DOESN'T HAVE A NUCLEUS OR ORGANELLES INSIDE. SINCE A EUKARYOTIC CELL HAS A NUCLEUS, AND A PROKARYOTIC CELL DOESN'T, THE REGULATION OF TRANSCRIPTION IS DIFFERENT BETWEEN THE TWO. IN A EUKARYOTE, THE MRNA THAT IS TRANSCRIBED IN THE NUCLEUS MUST PASS THROUGH THE NUCLEAR ENVELOPE TO BE TRANSLATED IN THE CYTOPLASM. BEFORE IT CAN LEAVE, IT HAS TO BE PROCESSED. SOME PARTS ARE ADDED TO THE STRAND, AND SOME ARE TAKEN OUT.

IN A PROKARYOTE, THERE'S NO NUCLEAR ENVELOPE, SO THE MRNA CAN BEGIN TRANSLATION RIGHT THERE IN THE CYTOPLASM. TRANSCRIPTION AND TRANSLATION OVERLAP WITH EACH OTHER. SO, THE PRODUCTION OF PROTEINS ACTUALLY BEGINS BEFORE THE MRNA STRAND IS COMPLETE. UNLIKE EUKARYOTES, PROKARYOTES HAVE MORE THAN ONE GENE ON AN MRNA STRAND. SO, WITH THE OVERLAP OF PROCESSES, ALL THE GENES ON THE MRNA END UP GETTING TRANSLATED TOGETHER. USUALLY, AN ORGANISM DOESN'T WANT TO TRANSLATE DIFFERENT PROTEINS AT THE SAME TIME BECAUSE DIFFERENT PROTEINS ARE INVOLVED IN DIFFERENT CELLULAR ACTIVITIES. SO, IN A PROKARYOTE, GENES THAT ARE RELATED TO EACH OTHER ARE FOUND SIDE-BY-SIDE ON THE ACTUAL DNA. CLUSTERS OF RELATED GENES ARE CALLED OPERONS. WHEN AN ENTIRE OPERON IS TRANSLATED, A WHOLE TEAM OF PROTEINS IS PRODUCED. EVERY PROTEIN ON THE TEAM CONTRIBUTES TO THE SAME CELLULAR FUNCTION.

A BROAD VIEW OF TRANSCRIPTIONAL REPRESSION INCLUDES CELLULAR PROCESSES THAT INTERFERE WITH THE TRANSCRIPTION OF GENES ON A GLOBAL OR LOCAL LEVEL. PROCESSES THAT DEGRADE, SEQUESTER, COVALENTLY MODIFY, OR REMOVE FROM NUCLEUS POSITIVELY ACTING TRANSCRIPTION FACTORS CAN CAUSE TRANSCRIPTIONAL REPRESSION OF GENES BY ELIMINATING AN ACTIVATING SIGNAL. RNA POLYMERASES AND BASAL FACTORS USED AT MOST PROMOTERS. IN THESE CASES, NEGATIVELY ACTING FACTORS NEED NOT DIRECTLY INTERACT WITH GENES TO INHIBIT TRANSCRIPTION. MANY FORMS OF TRANSCRIPTIONAL REPRESSION, HOWEVER, INVOLVE THE ACTIVITY OF DNA-BINDING PROTEINS THAT DO DIRECTLY CONTACT GENES. THE BINDING OF REPRESSORS CAN BE RELATIVELY NONSEQUENCE-SPECIFIC.

REGULATORY PROTEINS CAN WORK IN A CONTEXT-DEPENDENT MANNER, EITHER FACILITATING OR INHIBITING TRANSCRIPTION DEPENDING ON THE REGULATORY ELEMENT, SIGNALS FROM SIGNAL TRANSDUCTION CASCADES, RESPONDING BASAL PROMOTER, OR LEVELS OF COFACTORS

IN PROKARYOTES, LAC REPRESSOR LED TO AN INITIAL VIEW THAT MOST GENES WERE CONTROLLED BY REPRESSION. IN EUKARYOTES, EARLY STUDIES OF TRANSCRIPTIONAL REGULATION WERE BASED ON VIRAL GENE REGULATION. A NUMBER OF WELL-CHARACTERIZED VIRAL REGULATORY ELEMENTS FUNCTION AS DEDICATED TRANSCRIPTIONAL ACTIVATION ELEMENTS, LEADING TO A FOCUS ON TRANSCRIPTIONAL ACTIVATION. THE TERM TRANSCRIPTIONAL ENHANCER WAS COINED TO DESCRIBE A CISREGULATORY ELEMENT THAT FUNCTIONED TO ACTIVATE TRANSCRIPTION IN AN ORIENTATION AND DISTANCE INDEPENDENT MANNER. THESE ELEMENTS ARE OFTEN MORE COMPLEX, MEDIATING REPRESSION AS WELL AS ACTIVATION FUNCTIONS. IN MOST CASES, REPRESSOR-BINDING SITES ARE FOUND WITHIN REGIONS THAT ALSO CONTAIN BINDING SITES FOR ACTIVATORS, PRODUCING A CIS-REGULATORY ELEMENT WITH MULTIPLE POTENTIAL ACTIVITIES. DEDICATED NEGATIVELY ACTING ELEMENTS TERMED SILENCERS HAVE BEEN IDENTIFIED.

EUKARYOTIC CIS-REGULATORY ELEMENTS RARELY CONSIST OF SINGLE BINDING SITES FOR A TRANSCRIPTION FACTOR. INSTEAD, THEY GENERALLY COMPRISE SHORT SEGMENTS OF DNA TO WHICH A NUMBER OF PROTEINS BIND IN A SEQUENCE-SPECIFIC MANNER. SUCH ELEMENTS FORM NUCLEOPROTEIN COMPLEXES THAT CAN HAVE VARYING DEGREES OF HIGHER ORDER STRUCTURE . IN SOME CASES, THE DISTRIBUTION OF BINDING SITES FOR ACTIVATORS AND REPRESSORS REPRESENTS A CAREFULLY SELECTED DESIGN THAT REFLECTS HIGHLY COOPERATIVE INTERACTIONS BETWEEN THE FACTORS, WHILE IN OTHERS, BINDING SITE LOCATIONS ARE RELATIVELY FLEXIBLE, PERMITTING BINDING SITES TO DRIFT OVER THE COURSE OF EVOLUTIONARY TIME. DEPENDING ON THE RANGE OF ACTION, THE REPRESSION CAN BE LOCAL, LIMITED TO THE ENHANCER IN WHICH THE REPRESSOR BINDS, OR LONG-RANGE, WHICH RESULTS IN INHIBITION OF MULTIPLE ENHANCERS. REPRESSOR SITES MAY BE LOCATED CLOSE TO THE TRANSCRIPTIONAL INITIATION SITE, A FACT THAT MAY BE IMPORTANT IF REPRESSORS COMPETE WITH COMPONENTS OF THE BASAL MACHINERY FOR BINDING TO DNA. IN OTHER CASES, REPRESSORS

COVALENT MODIFICATION OF TRANSCRIPTIONAL ACTIVATORS AS DISCUSSED ABOVE, MODIFICATION, DEGRADATION OR SEQUESTRATION OF TRANSCRIPTIONAL ACTIVATORS CAN LEAD TO REPRESSION OF GENES. EXAMPLES OF SUCH MODIFICATIONS ARE INDICATED IN TABLE 2. IN MANY CASES, THE MODIFICATION CAN OCCUR WHEN THE FACTOR IS NOT BINDING TO A GENE AND/OR IS NOT LOCATED WITHIN THE NUCLEUS, OR ALTERNATIVELY, MODIFICATION CAN ALSO OCCUR TO DNA-BOUND TRANSCRIPTIONAL ACTIVATORS. THE PROTEINS THAT CARRY OUT SUCH MODIFICATIONS GENERALLY ARE NOT DNA-BINDING PROTEINS THEMSELVES, BUT ARE TARGETED TO ACTIVATORS VIA PROTEIN–PROTEIN INTERACTIONS. CONSTITUTIVE PATHWAYS THAT REMOVE COVALENT MODIFICATIONS CAN PROVIDE A LEVEL OF NEGATIVE REGULATION. THE SMAD PROTEINS, DOWNSTREAM EFFECTORS OF TRANSFORMING GROWTH FACTOR (TGF)-B PATHWAY SIGNALING, ARE PHOSPHORYLATED IN RESPONSE TO PATHWAY ACTIVATION, AND DEPHOSPHORYLATION RETURNS THEM TO AN INACTIVE STATE (INMAN ET AL. 2002). MAJOR PATHWAYS FOR DNA-BOUND REPRESSORS AND COREPRESSORS ARE: (1) DIRECT COMPETITION BETWEEN A REPRESSOR AND AN ACTIVATOR FOR THE SAME OR OVERLAPPING BINDING SITES; (2) INTERACTIONS WITH THE BASAL MACHINERY, INCLUDING RNA POLYMERASE II AND ASSOCIATED FACTORS, TO INHIBIT INITIATION OR ELONGATION; (3) DIRECT INTERACTION BETWEEN (CO)-REPRESSORS AND ACTIVATORS, RESULTING IN INHIBITION OF ACTIVATOR FUNCTION, SOMETIMES TERMED MASKING; AND (4) CHROMATIN ALTERATIONS, INVOLVING THE CHANGE OF DNA/NUCLEOSOMAL STRUCTURE, OR COVALENT MODIFICATION OF CHROMATIN OR DNA (FIG. 3). ADDITIONAL, LESS UNDERSTOOD MECHANISMS HAVE ALSO BEEN SUGGESTED, INCLUDING INTRANUCLEAR TARGETING OF REPRESSED GENES TO HETEROCHROMATIC SITES, REPULSION OF COACTIVATORS, OR POSSIBLE DIRECT PROTEOLYSIS BY TRANSCRIPTIONAL REPRESSORS (HE

PROKARYOTIC REPRESSION: SOME TRANSCRIPTION FACTORS FUNCTION TO REPRESS TRANSCRIPTION, WHILE OTHERS ACTIVATE TRANSCRIPTION. STILL OTHERS FUNCTION AS EITHER ACTIVATORS OR REPRESSORS, OFTEN ACCORDING TO THE POSITIONING TO THE Σ-FACTOR BINDING SITE IN THE TARGET PROMOTER THE BINDING AND RELEASE OF REPRESSORS AND ACTIVATORS THEMSELVES ARE OFTEN CONTROLLED BY COFACTOR BINDING. COFACTORS ARE MOLECULES THAT CAN RANGE WIDELY IN SIZE AND NATURE, FROM SMALL IONS, NUCLEOTIDES, COVALENTLY ATTACHED PHOSPHATE MOIETIES, AND SUGARS TO PEPTIDES OR WHOLE PROTEINS. ALTHOUGH MOST ACTIVATORS FUNCTION BY FIRST BINDING TO THE PROMOTER DNA BEFORE INTERACTING WITH RNAP, SOME ACTIVATORS (SUCH AS E. COLI MARA AND SOXS) ALSO BIND TO FREE RNAP IN THE CYTOSOL PRIOR TO BINDING THEIR TFBS.

THERE ARE FOUR MAIN MODES IN WHICH TFS HAVE BEEN DESCRIBED TO MEDIATE REPRESSION BY STERIC HINDRANCE, OFTEN BY BINDING OF THE REPRESSOR BETWEEN OR ON THE CORE PROMOTER ELEMENTS; (II) REPRESSION BY BLOCKING OF TRANSCRIPTION ELONGATION, OFTEN BY BINDING AT THE START OF THE CODING REGION (ROADBLOCK MECHANISM); (III) REPRESSION BY DNA LOOPING, WITH BINDING SITES OFTEN BOTH UPSTREAM AND DOWNSTREAM OF THE CORE PROMOTER (IN THIS CASE, AN INTERACTION BETWEEN TWO MONOMERS OF THE SAME TF IS POSSIBLE ONLY IF BOTH TFBSS ARE SPACED CORRECTLY); AND (IV) REPRESSION BY THE MODULATION OF AN ACTIVATOR. IN THE LATTER CASE, A REPRESSOR BINDS TO A TFBS THAT (PARTLY) OVERLAPS A DIFFERENT TFBS OF AN ACTIVATOR. THE BINDING OF THE REPRESSOR TO ITS SITE WILL THEN PREVENT THE BINDING OF THE ACTIVATOR TO ITS RESPECTIVE TFBS. AN EXAMPLE OF SUCH AN INTERACTION IS THAT BETWEEN THE CYTR AND CRP.

SIMILARLY, FOUR MODES OF ACTIVATION BY TFS HAVE BEEN DESCRIBED CLASS I ACTIVATION, IN WHICH THE TF BINDS UPSTREAM OF THE CORE PROMOTER AND INTERACTS WITH THE FLEXIBLE Α-SUBUNIT OF RNAP; (II) CLASS II ACTIVATION, IN WHICH THE TF BINDS THE DNA DIRECTLY ADJACENT (MOSTLY UPSTREAM) TO THE CORE PROMOTER AND PROMOTES Σ-FACTOR BINDING; (III) ACTIVATION BY DNA CONFORMATIONAL CHANGE, IN WHICH THE TF BINDS TO THE CORE PROMOTER TO ENABLE IT TO BE BOUND BY A Σ-FACTOR, OFTEN BY TWISTING THE DNA HELIX; AND (IV) ACTIVATION BY THE MODULATION OF A REPRESSOR, ALLEVIATING THE REPRESSION EFFECT. AN EXAMPLE OF THE LATTER MODE (ALSO TERMED ANTIREPRESSION) WAS RECENTLY DISCOVERED FOR THE B. SUBTILIS COMPETENCE ACTIVATOR COMK, A MINOR GROOVE BINDING PROTEIN THAT BINDS ADJACENT TO THE REPRESSORS ROK AND CODY AT ITS OWN COMK PROMOTER. ALTHOUGH COMK BINDING TO THE DNA DOES NOT RESULT IN THE PHYSICAL DISPLACEMENT OF ROK AND CODY, IT REMOVES THE REPRESSION EFFECT AND THUS ACTIVATES THE EXPRESSION OF THE GENE