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Transcription

RNA polymerase molecules on DNA are producing ribosomal RNA molecules that bind to ribosomal proteins.

Transcription starts at a "promoter." Transcription ends at a "terminator."

A transcription unit = a gene or operon

Genes are tightly packed (some overlapped) in bacterial genomes but loosely packed with many long intergenic spaces in eukaryotic genomes.

Bubble

Bubble

Transcription cycle
R Y +1 R

Intrinsic terminators code for RNA hairpin - oligo(U)

Promoter for E. coli 70-RPase Holoenzyme (core + ) finds and binds a promoter.
Termination complex Close complex

RNA is released in termination and RPase dissociates from DNA


Elongation is processive and stable.

interacts with the -35 and -10 hexamers. Isomerization from closed to open binary complex A region around -10 and +1 is unwound to form a bubble. Nucleotides bind to form ternary complex Short RNAs are produced, as the contact with -35 is lost.

Elongation complex

Open complex Abortive initiation cycling

is released and promoter is cleared.

Short RNAs are repetitively released and new RNAs are produced.

Eukaryotic transcription

Nuclear RPase I, II, III and IV (?), Mitochondrial and chloroplast RPases

A RPase II promoter

TFIID binds and bends TATA box.

Many initiation factors are loaded.


Initiation complex

by TFIIH during Abortive cycling TFIID remains at promoter and PRPase moves down.

Many proteins (>100 subunits) must assemble around the +1 site. The order of assembly varies from gene to gene. Some protein complexes are preassembled before brought to DNA together.

Eukaryotic mRNA processing in nucleus


5'-UTR 3'-UTR

Capping at the 5' end of mRNA

Exons + Introns All the mRNA processing events, capping, splicing, 3'-end processing occur during transcription within nucleus. The processing factors bind to CTD of RPase and act on the mRNA parts that are coming out of the RPase. Only RPase II has CT 5'-P-5' linkage CBC (cap binding complex) marks a successfully capped end.

Eukaryotic mRNA splicing


Two sequential transesterification reactions at a site Exon = Expressed part, Intron = Intervening part
5'-Splice site Branch site 3'-Splice site

EJC (exon junction complex) marks a successfully spliced site

The 5'-splice, branch and 3'-splice sites are highly homologous in all splicings. Splicing occurs sequentially as soon as CTD-bound splicing factors recognize relevant sites sequentially coming out of RPase II. Thus, a mature RNA of exon3-exon1-exon2 cannot be made in vivo.

Spliceosome: snRNA-snRNA interactions

SR proteins mark exons.

RNA-RNA Rearrangements using ATP hydrolysis.

Alternative and abnormal splicing

Alternative splicing occurs by skipping exons. Diverse proteins are made from alternatively spliced mRNAs, which all are made from a gene. Other forms of diversity are alternative start sites of transcription and translation. Many of these are subject to regulation. Abnormal splicing can occur by some mutations or polymorphisms in DNA that inactivate normal splice sites or activate cryptic splice sites. Some are associated with diseases.

Self-splicing
Two sequential transesterification reactions at a site

Self-splicing RNA has to fold into a proper structure without the help of spliceosome. Thus, most of the intron sequences are critical in selfsplicing, whereas they are not critical in spliceosome splicing.

Processing at 3' end and nuclear transport 3'-Cleavage and polyadenylation


Poly(A) polymerase does not require a template. After 3'-cleavage, RPase II continues for some distance. Poly(A)-binding proteins mark polyadenylation. Nuclear export receptor is loaded on mature mRNA. RNA debris are degraded by nuclear exosome.
Nuclear transport "export-ready nuclear pore RNA complex
CPSF CstF

Non-coding RNA synthesis and processing in nucleolus


In nucleolus, pre-rRNA is produced by RPase I, modified, cleaved into 3 rRNAs, and complexed with ribosomal proteins. (5S rRNA is produced by RPase III and not modified.) Subnuclear structures

In nucleolus, some ribonucleoprotein complexes (RNAs + proteins) are assembled (e.g. ribosomal subunits, U6 snRNP, telomerase, signal recognition particle, etc.) and tRNAs are processed. snoRNA (encoded by introns) and snRNA produced mostly by RPase II are modified at Cajal bodies and GEMS.

Figure 6-47. The function of the nucleolus in ribosome and other ribonucleoprotein synthesis.

Terms:
CTD carboxyl terminal domain SR proteins -serine/arginine rich proteins hnRNPs -heterogeneous nuclear ribonuclear proteins CstF -cleavage stimulation factor F CPSF -cleavage and polyadenylation specificity factor

Additional subnuclear structures: 1.Cajal bodies (named for scientist who first described them in 1906) 2.GEMS (Gemini of coiled bodies) -resemble one another and are frequently paired in the nucleus -may be sites where snRNAs and snoRNAs undergo their final modifications and assembly with protein -also sites where the snRNPs are recycled and their RNAs are reset after the rearrangements that occur during splicing 3. interchromatin granule clusters (also called speckles) -stockpiles of fully mature snRNPs that are ready to be used in splicing of pre-mRNAs

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