Chapter 10

Transcription

RNA processing

Translation

Jones and Bartlett Publishers © 2005

Terms (mRNA)

• primary transcript

• coding sequence (open reading frame, ORF)

• prokaryotic mRNA = primary transcript

• Eukaryotic transcripts are converted into mRNA through RNA processing:– Modification of the 5’ end– Extension of 3’ end– Excision of untranslated embedded sequences.

The Central Dogma

Polycistronic messages

• In bacteria and eukaryotic organelles, sometimes more than one polypeptide is encoded in a single message.

• These are called polycistronic mRNAs.

Polycistronic mRNAs

Eukaryotic Transcription

• In eukaryotes, three different polymerases are used.

• No polycistronic mRNAs are produced.

(All eukarytoic mRNAs are monocistronic.)

• The mRNA molecules are chemically modified in eukaryotes.

Eukaryotic RNA Polymerases

Enzyme Cellular location ProductRNA polymerase I Nucleolus rRNA

RNA polymerase II Nucleus mRNAsnRNA

RNA polymerase III Nucleus tRNA,5S

RNA, snRNA

Promoters for eukaryotic genes

• There is more variability in promoters and termination sequences.

• TATA box : 5’-TATAAA-3’, -25 bp, with GC regions near it.

• CAAT box: GCCAATCT, -80 bp

• GC box: GGGCGC, multiple copies.

Promoters in eukaryotes

Modification of primary transcripts

• Three major alterations to mRNA:

–Modified guanosine cap at 5’ end

– 3’ end gets a poly(A) tail.

– pre-mRNA is processed to remove specific internal sequences by splicing them out.

5’ cap in eukaryotes

Polyadenylation

• The poly(A) tail is added by poly(A) polymerase.

• About 200 adenosines are added to the 3’ end of the primary transcript.

• Poly(A) tails help geneticists isolate mRNAs from cells.

RNA Splicing

• In the 1960s, it was observed that RNA molecules were larger than predicted, based on protein structure.

• In 1977, internal, non-coding sequences were discovered.

• These internal, non-coding sequences are called introns.

Spliceosomes and the GU-AG rule

• Intron-Exon junctions have a conserved sequence that is recognized by an enzyme complex (spliceosome).

• The sequence is GU-AG, and is almost universal.

• A spliceosome has nucleic acids, 8-10 proteins, and snRNAs.

RNA processing

Transcription and RNA processing are coupled processes.

Molecular details of the splicing of an intron

Introns begin with a GU and

end with an AG. There is also an

internal A nucleotide where the

branch junction is formed.

The spliced intron is known as a lariat (with a loop and tail).

Splicing of introns is mediated by small nuclear ribonucleoprotein particles (snRNPs)

In this diagram,

only the RNA component of the snRNPs

is shown. Base pairing

between snRNA and the intron is involved in the splicing

reaction.

DNA-RNA hybridization showing the parts of DNA that are missing from the mature mRNA and the part of the mRNA (polyA tail) missing in the DNA

Mutation in a splice site may result in the retention of the entire (or part) of an intron

Exon-shuffle model

• Introns may play a role in gene evolution.

• In some proteins, each exon has its own independent folding characteristics.

• Folding domains (=exons) can be grouped together to give new proteins with new functions.

• This is called the exon-shuffle model.

• Not all genes have domain boundaries that correlate to exons.

Transcription occurs in the nucleus, as does RNA processing.

Export to the cytoplasm happens before translation occurs.

Translation of mRNA into amino acid sequences

• The genetic code consists of three bases.

• A codon is the ‘code word’ for each amino acid.

• 4 x 4 x 4 =64, but only 20 amino acids are used in proteins, so the genetic code is said to be degenerate.