Initiation of rDNA transcription Two transcription factors, UBF and SL1, bind cooperatively to the rDNA promoter and recruit RNA polymerase I to form an initiation complex. One subunit of SL1 is the TATA-binding protein (TBP) and the other is TBP- associated Factor (TAF).

Transcription-control elements in genes transcribed by RNA polymerase I (a) and III (b,c). Assembly of transcription-initiation complexes on these genes begins with the binding of specific general transcription factors to the control elements

Initiation of transcription by RNA polymerase I. One possible model envisages initial binding of the two identical subunits of the upstream binding factor to the upstream control element and the core promoter element, and forcing these two sequences to come into close proximity, enabling their subsequent binding by the selectivity factor 1 (SL1) which consists of four subunits. The stabilized structure permits subsequent binding of other factors (not shown) and subsequently RNA polymerase I.

Examples of chemical modifications occurring with nucleotides in rRNA and tRNA

Modification Details Example

Methylation Addition of one or more -CH3

groups to the base or sugar

                 Methylation of guanosine gives 7-methylguanosine

Deamination Removal of an amino (-NH2)

group from the base

              Deamination of adenosine gives inosine

Sulfur substitution Replacement of oxygen with sulfur

           4-Thiouridine

Base isomerization Changing the positions of atoms in the ring component of the base

           Isomerization of uridine gives pseudouridine

Double-bond saturation Converting a double bond to a single bond

            Double bond saturation converts uridine to dihydrouridine

The chemical modification and nucleolytic processing of a eucaryotic 45S precursor rRNA molecule into three separate ribosomal RNAs. As indicated, two types of chemical modifications are made to the precursor rRNA before it is cleaved. Nearly half of the nucleotide sequences in this precursor rRNA are discarded and degraded in the nucleus. The rRNAs are named according to their “S” values, which refer to their rate of sedimentation in an ultra-centrifuge. The larger the S value, the larger the rRNA.

Modifications of the precursor rRNA by guide RNAs. (A) Two prominent covalent modifications occur after rRNA synthesis; the differences from the initially incorporated nucleotide are indicated by red atoms. (B) As indicated, snoRNAs locate the sites of modification by base-pairing to complementary sequences on the precursor rRNA. The snoRNAs are bound to proteins, and the complexes are called snoRNPs. snoRNPs contain the RNA modification activities, presumably contributed by the proteins but possibly by the snoRNAs themselves.

Processing of pre-rRNA and assembly of ribosomes in eukaryotes. (a) Major intermediates and times required for various steps in pre-rRNA processing in higher eukaryotes. Ribosomal and nucleolar proteins associate with 45S pre-rRNA soon after its synthesis, forming an 80S pre-rRNP. Synthesis of 5S rRNA occurs outside of the nucleolus. The extensive secondary structure of rRNAs is not represented here. Note that RNA constitutes about two-thirds of the mass of the ribosomal subunits, and protein about one-third. (b) Pathway for processing of 6.6-kb (35S) pre-rRNA primary transcript in S. cerevisiae. The transcribed spacer regions (tan), which are discarded during processing, separate the regions corresponding to the mature 18S, 5.8S, and 25S rRNAs. All of the intermediates diagrammed have been identified; their sizes are indicated in red type.

Pre-rRNA processing in Escherichia coli. The pre-rRNA, containing copies of the 16S, 23S and 5S rRNAs, is cleaved by ribonucleases III, P and F, and the resulting molecules trimmed by ribonucleases M16, M23 and M5 to give the mature rRNAs. Eukaryotic pre-rRNAs, which contain the rRNA sequences in the order 18S-5.8S-28S, are processed in a similar fashion. Note that a

tRNA is located between the 16S and 23S sequences in the E. coli pre-rRNA.

Processing of ribosomal RNAs Prokaryotic cells contain three rRNAs (16S, 23S, and 5S), which are formed by cleavage of a pre-rRNA transcript. Eukaryotic cells (e.g., human cells) contain four rRNAs. One of these (5S rRNA) is transcribed from a separate gene; the other three (18S, 28S, and 5.8S) are derived from a common pre-rRNA. Following cleavage, the 5.8S rRNA (which is unique to eukaryotes) becomes hydrogen-bonded to 28S rRNA.

Figure 8.28. Ribosome assembly Ribosomal proteins are imported to the nucleolus from the cytoplasm and begin to assemble on pre-rRNA prior to its cleavage. As the pre-rRNA is processed, additional ribosomal proteins and the 5S rRNA (which is synthesized elsewhere in the nucleus) assemble to form preribosomal particles. The final steps of maturation follow the export of preribosomal particles to the cytoplasm, yielding the 40S and 60S ribosomal subunits.

Eukaryotic RNA polymerase 18S+ 30 proteins= 40S Poly merase I, II, III 5.8S, 5S, 28S+ 5 proteins = 60STranscription Factors UCE (upstream control element) 16S + 21 proteins = 30SUBF (upstream binding factor) 5S, 23S + 34 protein = 50SSL1 (TBP= TATA binding protein) (TAF= TBP associated factor)Initiation complexChemical modificationsMethylationPseudouridineSmall nucleolar RNA (sno RNA)Sno RNPProkaryotic pre-rRNA16S, 23S, 5S rRNALeader, Trailer, SpacerRnase IIIM16, M23, M5Eukaryotic pre-rRNA18S, 5.8S, 28S rRNA