Two critical events precede protein synthesis. First, aminoacyl-tRNA synthetases attach amino acids to their respective tRNAs with high specificity through a two-step reaction that begins with the activation of the amino acid using AMP, derived from ATP. Second, ribosomes must dissociate into their subunits at the conclusion of each translation cycle. In bacteria, this dissociation is actively facilitated by RRF and EF-G, while IF3 binds to the free 30S subunit, preventing its reassociation with the 50S subunit to form a complete ribosome.

The initiation codon in prokaryotes is typically AUG but can also be GUG or, more rarely, UUG. The initiating aminoacyl-tRNA is N-formyl-methionyl-tRNAfMet. N-formyl-methionine (fMet) is the first amino acid incorporated into a polypeptide chain, although it is often removed during protein maturation. The 30S initiation complex is formed by the association of a free 30S ribosomal subunit with mRNA and fMet-tRNAfMet. This binding depends on base pairing between the Shine-Dalgarno sequence, located just upstream of the initiation codon in mRNA, and a complementary sequence at the 3'-end of the 16S rRNA. IF3 mediates this interaction with the assistance of IF1 and IF2, which are all bound to the 30S subunit at this stage.

IF2 plays a central role in promoting the binding of fMet-tRNAfMet to the 30S initiation complex, while the other two initiation factors provide essential support. GTP is required for IF2 binding under physiological IF2 concentrations, though it is not hydrolyzed during this process. The complete 30S initiation complex consists of one 30S ribosomal subunit, one molecule each of mRNA, fMet-tRNAfMet, GTP, IF1, IF2, and IF3. GTP hydrolysis occurs after the 50S subunit joins the 30S complex to form the functional 70S initiation complex.

Ribosomal RNAs are synthesized in the nucleoli of eukaryotic cells as precursors that require processing to yield mature rRNAs. The sequence of RNAs in the precursor is universally 18S, 5.8S, and 28S across all eukaryotes, although the precise sizes of the mature rRNAs differ among species. In human cells, the precursor is 45S, which undergoes a processing scheme that produces 41S, 32S, and 20S intermediates, with snoRNAs playing crucial roles in these steps.

Extra nucleotides are removed from the 5'-ends of pre-tRNAs in a single step through endonucleolytic cleavage catalyzed by RNase P. Both bacterial and eukaryotic RNase P enzymes have a catalytic RNA subunit called M1 RNA. In E. coli, RNase II and polynucleotide phosphorylase cooperate to remove most of the additional nucleotides at the 3'-end of a tRNA precursor but halt at the 12-base stage. RNases PH and T are primarily responsible for removing the last two nucleotides. In eukaryotes, a single enzyme, tRNA 3'-processing endoribonuclease (3'-tRNase), performs the processing of the 3'-end of a pre-tRNA.

Trypanosome mRNAs are generated through trans-splicing, which links a short leader exon with one of many independent coding exons. In trypanosomatid mitochondria, incomplete mRNAs require editing before translation. Editing occurs in the 3'→5' direction through sequential actions of one or more guide RNAs (gRNAs). These gRNAs bind to unedited mRNA regions, providing A's and G's as templates for inserting missing U's or deleting extra U's.

In higher eukaryotes, including fruit flies and mammals, some adenosines in mRNAs must be post-transcriptionally deaminated to inosine for correct translation. This type of RNA editing is performed by enzymes called adenosine deaminases acting on RNAs (ADARs). Additionally, certain cytidines must be deaminated to uridine for accurate mRNA coding. Post-transcriptional gene regulation often involves such modifications to ensure proper gene expression.

Capping occurs in several steps: initially, RNA triphosphatase removes the terminal phosphate from pre-mRNA. Subsequently, guanylyl transferase adds the capping GMP derived from GTP, followed by two methyl transferases that methylate the N7 position of the capping guanosine and the 2'-O-methyl group of the penultimate nucleotide. These processes take place early in transcription, before the RNA chain exceeds 30 nucleotides in length. The cap plays a crucial role in ensuring proper splicing of some pre-mRNAs, facilitating the transport of mature mRNAs out of the nucleus, protecting mRNA from degradation, and enhancing its translatability.

Most eukaryotic mRNAs and their precursors possess a poly(A) tail approximately 250 nucleotides long at their 3'-ends, added post-transcriptionally by poly(A) polymerase. The poly(A) tail increases both the stability and translatability of the mRNA, with the relative importance of these effects differing across systems.

Transcription of eukaryotic genes beyond the polyadenylation site, after which the transcript is cleaved and polyadenylated at the newly formed 3'-end. An efficient mammalian polyadenylation signal includes an AAUAAA motif about 20 nucleotides upstream of the polyadenylation site, followed 23–24 base pairs later by a GU-rich sequence and then a U-rich motif. Variations in these sequences influence polyadenylation efficiency, with plant signals allowing more flexibility around the AAUAAA motif than animal signals, and yeast signals rarely containing the AAUAAA motif.

Polyadenylation involves both cleavage of the pre-mRNA and the addition of the poly(A) tail at the cleavage site. The cleavage process requires multiple proteins, including CPSF, CstF, CF I, CF II, poly(A) polymerase, and the CTD of the largest subunit of RNA polymerase II. Among these, CPSF-73 is responsible for cleaving the pre-mRNA.

Nuclear mRNA precursors undergo splicing through a lariat-shaped or branched intermediate. In addition to the consensus sequences at the 5′ and 3′ ends of nuclear introns, branchpoint consensus sequences are also present. In yeast, this sequence is almost invariant as UACUAAC, whereas in higher eukaryotes, the consensus sequence is more variable, represented as YNCURAC. In all cases, the branched nucleotide corresponds to the final A in the sequence. The yeast branchpoint sequence also determines which downstream AG serves as the 3′ splice site.

Splicing occurs on a complex structure known as the spliceosome. Yeast and mammalian spliceosomes have sedimentation coefficients of approximately 40S and 60S, respectively. Genetic studies have revealed that base pairing between U1 snRNA and the 5′ splice site an mRNA precursor is necessary but not sufficient for splicing. The U6 snRNP also forms a base-pairing association with the 5′ end of the intron, which begins before the formation of the lariat intermediate but may alter its nature after this initial step. This interaction between U6 and the splicing substrate is critical for the splicing process. Furthermore, U6 interacts with U2 during splicing.

The U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint, an interaction essential for splicing. Additionally, U2 forms significant base pairs with U6 to create a region referred to as helix I, which plays a role in aligning these snRNPs for the splicing process. The U4 snRNA base-pairs with U6, contributing to the splicing mechanism.

Eukaryotic DNA associates with basic protein molecules called histones to form nucleosomes. Each nucleosome consists of four pairs of histones (H2A, H2B, H3, and H4) arranged in a wedge-shaped disc, around which 146 base pairs (bp) of DNA are wrapped. Histone H1, which is not part of the core nucleosome, is more easily removed from chromatin than the core histones.

In the second level of chromatin folding, both in vitro and presumably in vivo, a string of nucleosomes forms a 30-nanometer (nm) fiber. Studies indicate that this fiber exists in at least two forms within the nucleus: inactive chromatin, characterized by a high nucleosome repeat length (approximately 197 bp), tends to adopt a solenoid folding structure and interacts with histone H1, which stabilizes its structure. Conversely, active chromatin, with a lower nucleosome repeat length (around 167 bp), folds according to the two-start double helical model.

The third level of chromatin condensation involves the formation of radial loop structures in eukaryotic chromosomes. The 30-nm fiber forms loops ranging from 35 to 85 kilobases () in length, anchored to the chromosome's central matrix.

Core histones (H2A, H2B, H3, and H4) assemble nucleosome cores on naked DNA. Transcription of a class II gene in reconstituted chromatin, with an average of one nucleosome core per 200 bp of DNA, shows approximately 75% repression compared to naked DNA. The remaining 25% activity is attributed to promoter sites not covered by nucleosome cores. Histone H1 further represses template activity beyond the core nucleosomes. This repression can be mitigated by transcription factors, some of which, like Sp1 and GAL4, act as both antirepressors (preventing repression by histone H1) and transcription activators. Others, such as the GAGA factor, function solely as antirepressors, likely competing with histone H1 for binding.

Eukaryotic activators consist of at least two domains: a DNA-binding domain and a transcription-activating domain. DNA-binding domains include motifs such as zinc modules, homeodomains, bZIP, or bHLH motifs. Transcription-activating domains can be acidic, glutamine-rich, or proline-rich. Zinc fingers are characterized by an antiparallel β-sheet followed by an α-helix. The β-sheet contains two cysteines, and the α-helix contains two histidines, which coordinate with a zinc ion to form the finger-shaped structure. This coordination facilitates specific recognition of the DNA target within the major groove.

The DNA-binding motif of the GAL4 protein includes six cysteines that coordinate two zinc ions in a bimetal thiolate cluster. This motif features a short α-helix that extends into the DNA major groove, forming specific interactions. Additionally, the GAL4 monomer contains an α-helical dimerization motif that forms a parallel coiled coil with the α-helix of another GAL4 monomer.

Type I nuclear receptors are located in the cytoplasm, bound to other proteins. Upon binding their hormone ligands, these receptors release their cytoplasmic partners, translocate to the nucleus, bind to enhancers, and function as activators. A representative example is the glucocorticoid receptor, which contains a DNA-binding domain with two zinc modules. One module provides DNA-binding residues in a recognition α-helix, while the other facilitates protein-protein interactions for dimer formation. These zinc modules use four cysteine residues to complex the zinc ion, unlike classical zinc fingers, which use two cysteines and two histidines.

Homeodomains in eukaryotic activators contain a DNA-binding motif that operates similarly to the helix-turn-helix motifs in prokaryotes, where a recognition helix fits into the DNA major groove.

Transcription factors bind to class II promoters in vitro in the following sequence: (1) TFIID, with assistance from TFIIA, attaches to the TATA box. (2) TFIIB binds subsequently. (3) TFIIF facilitates the binding of RNA polymerase II. The remaining factors bind in this order:IIE and TFIIH, creating the DABPolFEH preinitiation complex. Notably, TFIIA's involvement appears to be optional in vitro.

TFIID is composed of a TATA-box-binding protein (TBP) and 13 additional polypeptides referred to as TBP-associated factors (TAFs). The TATA-box-binding domain of TBP is located within its C-terminal 180 amino acid fragment. The interaction between TBP and the TATA box occurs within the DNA minor groove. The saddle-like shape of TBP aligns with the DNA, and the underside of the "saddle" forces the minor groove open, bending the TATA box by approximately 80 degrees. TBP is essential for the transcription of most genes across all three classes, not limited to class II genes.

Many TAFs are evolutionarily conserved across eukaryotes and serve multiple roles, including interacting with core promoter elements and gene-specific transcription factors. TAF1 and TAF2 enable TFIID to bind to initiator elements and downstream promoter elements (DPEs), allowing TBP to bind to certain TATA-less promoters. TAF1 and TAF4 facilitate TFIID's interaction with Sp1 bound to GC boxes upstream of the transcription start site, ensuring TBP binding to TATA-less promoters containing GC boxes. Different TAF combinations are required to respond to various transcription activators, particularly in higher eukaryotes. Additionally, TAF1 exhibits enzymatic activity as both a histone acetyltransferase and a protein kinase. However, TFIID is not universally required in higher eukaryotes. For instance, some Drosophila promoters require an alternative factor, TRF1, while others depend on a TBP-free TAF complex.

Eukaryotic nuclei house three distinct RNA polymerases, which can be separated using ion-exchange chromatography. RNA polymerase I resides in the nucleolus, while other two are located in the nucleoplasm. Each of these polymerases performs specific transcriptional roles. Polymerase I synthesizes a large precursor to the major rRNAs (5.8S, 18S, and 28S in vertebrates). Polymerase II generates hnRNAs, precursors to mRNAs, as well as miRNA precursors and most small nuclear RNAs (snRNAs). Polymerase III is responsible for producing precursors of 5S rRNA, tRNAs, and various other small cellular and viral RNAs.

The subunit structures of the three nuclear polymerases have been analyzed in several eukaryotes, revealing multiple subunits, including two large ones exceeding 100 kD in molecular mass. Common subunits appear in all three polymerases across eukaryotes. In yeast, the genes encoding all 12 RNA polymerase II subunits have been sequenced and subjected to mutation analysis. Among these subunits, three resemble the core subunits of bacterial RNA polymerases in structure and function, five are shared by all three nuclear polymerases, two are dispensable under normal conditions, and two do not fit into these categories.

Subunit IIa, the primary product of the yeast RPB1 gene, can be converted to IIb in vitro through the proteolytic removal of the carboxyl-terminal domain (CTD), which consists of repeated heptapeptides. In vivo, subunit IIa is phosphorylated at two serines within the CTD heptad to form IIo. The enzyme containing the IIa subunit (polymerase IIA) binds to the promoter, while the enzyme with the IIo subunit (polymerase IIO) participates in transcript elongation.

The structure of yeast pol II D4/7 reveals a deep cleft capable of accommodating a DNA template. The catalytic activity and functional mechanisms of these polymerases underscore their critical roles in eukaryotic transcription.

The repressors of the λ-like phages possess recognition helices that fit sideways into the major groove of the operator DNA. Specific amino acids on the DNA-facing side of the recognition helix establish precise contacts with bases in the operator, and these interactions determine the specificity of the protein-DNA binding. Altering these amino acids can modify the specificity of the repressor. Both the λ repressor and the Cro protein exhibit affinity for the same operators, but their microspecificities for OR1 or OR3 are defined by interactions between distinct amino acids in the recognition helices of the two proteins and the base pairs in the respective operators.

The cocrystal structure of a λ repressor fragment bound to an operator fragment provides detailed insight into the protein-DNA interactions. The most critical contacts occur in the major groove, where amino acids on the recognition helix, along with other amino acids, form hydrogen bonds with the edges of DNA bases and the DNA backbone. Some of these hydrogen bonds are reinforced by hydrogen bond networks involving two amino acids and multiple sites on the DNA. The structural data derived from the cocrystal closely align with prior biochemical and genetic findings.

X-ray crystallography of a phage 434 repressor fragment/operator-fragment complex reveals probable hydrogen bonding between amino acid residues in the recognition helix and base pairs in the repressor. It also indicates a potential van der Waals interaction between an amino acid in the recognition helix and a base in the operator. The DNA in the deviates significantly from its typical regular shape, bending slightly to facilitate the necessary base/amino acid contacts. Additionally, the central region of the helix, the two half-sites, is wound more tightly, while the outer regions are wound more loosely than usual. These structural deviations are supported by the base sequence of the operator.

Bacteria undergo significant shifts in transcription patterns during various processes, such as phage infection or sporulation, and have evolved multiple mechanisms to facilitate these changes. For instance, the transcription of phage SPO1 genes in infected B. subtilis cells follows a temporal sequence, where early genes are transcribed first, followed by middle genes, and finally late genes. This transition is regulated by phage-encoded sigma factors that associate with the host's core RNA polymerase and alter its specificity from early to middle to late genes. The host sigma factor is specific to the phage early genes, while the phage gp28 protein changes the specificity to middle genes, and gp33 and gp34 proteins direct specificity to late genes.

When B. subtilis undergoes sporulation, an entirely new set of sporulation-specific genes is activated, while many vegetative genes are turned off. This switch primarily occurs at the transcriptional level and is mediated by several new sigma factors that displace the vegetative sigma factor from the core RNA polymerase, redirecting transcription to sporulation-specific genes. Each sigma factor recognizes its own preferred promoter sequence.

Certain prokaryotic genes must be transcribed under conditions where two different sigma factors are active. These genes are equipped with dual promoters, each recognized by one of the sigma factors, ensuring their expression regardless of which factor is present and enabling differential regulation under varying conditions. For example, in E. coli, the heat shock response and responses to low nitrogen and starvation stress are regulated by alternative sigma factors—sigma32 (σH), sigma54 (σN), and sigma38 (σS)—which replace the primary sigma factor sigma70 (σA) and direct RNA polymerase to alternative promoters. Additionally, many sigma factors are regulated by anti-sigma factors that bind to specific sigma factors and inhibit their interaction with the core RNA polymerase. Some of these anti-sigma factors are further regulated by additional mechanisms.