Chapter 14 – RNA molecules and RNA processing

Gene organization • Francis Crick – 1958

– Nucleotide sequence of a gene directly codes for amino acid sequence of polypeptide

• Gene contains interruptions of nucleotides that do not code for amino acids– Eukaryotic genes

Gene organization • DNA and RNA transcripts

within the nucleus are larger than transcripts found in the cytoplasm– Exons are coding regions

– Introns are intervening sequences in eukaryotic genes

• Occasionally seen in prokaryotes as well

• Are spliced out of pre-mRNA/primary transcript before leaving the nucleus

Messenger RNA (mRNA)• Structure

– 5′ untranslated region• Bacteria contain Shine-

Dalgarno sequence– Serves as ribosome

attachment site

– Protein coding region• Codes for amino acids

• Codon – 3 nucleotide sequence that codes for one amino acid

– 3′ untranslated region• Aids in stability of molecule

Prokaryotic mRNA

• Since introns are rare, mRNA can begin to be translated before transcription is complete– Ribosome associates with Shine-Dalgarno

sequence, and moves down mRNA molecule in 5′→3′ direction

Eukaryotic mRNA• mRNA requires post-

transcriptional modifications before exiting nucleus to cytoplasm (site of translation)

• 5′ cap– Guanine nucleotide added 5′

to 5′– Several nucleotides at the 5′

end are methylated– Stabilizes molecule; aids in

initiation of translation

Post-transcriptional modifications cont

• polyA tail

– At 3′ end there is at least one possible cleavage site where nucleotides are removed

• After removal, 50-250 adenine nucleotides are added

– Polyadenylation

– With associated proteins, stabilizes molecule

Post-transcriptional modifications cont

• RNA splicing– Splicing out of introns

requires 5′ splice site, 3′ splice site, and branch point

– Spliceosome• 5 snRNPs and several

other proteins

– Process of splicing• 5′ end of intron is cut

and folds back on itself to attach to branch point sequence

– Forms a lariat• 3′ end of intron is cut

and intron is released• 3′ of exon #1 is ligated to

5′ end of exon #2• Intron reverts to linear

form and is degraded

Alternative processing • Approx. 30,000 genes

in genome; 120,000 polypeptides– A single pre-mRNA

molecule can give rise to different mature mRNA – each results in a different polypeptide

• Alternative splicing– In addition to intron

removal, exons may be removed as well

Alternative processing cont• Multiple 3′

cleavage sites

– Cleavage may occur at different sites before polyA tail is added

– Any exons not included will yield a different polypeptide

Transfer RNA (tRNA)

• 74-95 nucleotides long

• Can have chemically modified bases in addition to the normal 4 normally present in RNA

tRNA cont• Complementary base pairs

form a cloverleaf shape (folds into an “L” 3D)

• 3′ end is the acceptor arm – where a specific amino acid attaches

• Anticodon arm contains 3 nucleotides (anticodon) that recognize codon of mRNA

• Initial transcript contains introns that are removed

Ribosomal RNA (rRNA)

• Original transcript for ribosomal RNA is cleaved by snoRNA– snoRNA + proteins form snoRNPs

• Sizes of rRNA measured in Svedberg untis (S) – how fast substances sediment out in a centrifugal field)– Based on molecular weight and structure – not cumulative

• Ribosomes– 1 or more molecules of rRNA and approx 50 proteins– Complete ribosome consists of 2 subunits – large and

small

RNA interference (RNAi)• miRNA (microRNA) and

siRNA (small interfering RNA)

• Both arise from double-stranded RNA, which is cute by enzyme Dicer – fragments are miRNA and siRNA

• Small fragments bind to mRNA– miRNA inhibits translation – siRNA – degrades mRNA