While bacteria use a single type of RNA polymerase for transcription, eukaryotic cells employ three: RNA polymerase I, RNA polymerase II, and RNA polymerase III. These polymerases are responsible for transcribing different types of genes. RNA polymerases I and III transcribe the genes encoding transfer RNA, ribosomal RNA, and various other RNAs that play structural and catalytic roles in the cell. RNA polymerase II transcribes the rest, including all those that encode proteins—which constitutes the majority of genes. in eukaryotes

Whereas the bacterial RNA polymerase (along with its sigma subunit) is able to initiate transcription on its own, eukaryotic RNA polymerases require the assistance of a large set of accessory proteins. Principal among these are the general transcription factors, which must assemble at each promoter, along with the polymerase, before transcription can begin.

The mechanisms that control the initiation of transcription in eukaryotes are much more elaborate than those that operate in prokaryotes. In bacteria, genes tend to lie very close to one another, with only very short lengths of nontranscribed DNA between them. But in plants and animals, including humans, individual genes are spread out along the DNA, with stretches of up to 100,000 nucleotide pairs between one gene and the next. This architecture allows a single gene to be controlled by a large variety of regulatory DNA sequences scattered along the DNA, and it enables eukaryotes to engage in more complex forms of transcriptional regulation than do bacteria.

Eukaryotic transcription initiation must deal with the packing of DNA into nucleosomes and higher-order forms of chromatin structure.