The big picture

• DNA -> RNA -> protein
• The first step is to make RNA
• Process is similar to DNA synthesis (polymerases, 5' to 3', etc)
• • Except: No proofreading, no primer. U replaces T

Four types of RNA

• mRNA
• • Messenger RNA, encodes the amino acid sequence of a polypeptide
• rRNA
• • Ribosomal RNA, forms complexes called ribosomes with protein, the structure on which mRNA is translated
• tRNA
• • Transfer RNA, transports amino acids to ribosomes during translation
• snRNA
• • Small nuclear RNA, forms complexes with proteins used in eukaryotic RNA processing

Basic process of transcription

• Ingredients include:
• DNA template, RNA Polymerase, NTPs, MgCl2; Primer not necessary
• Read the template DNA 3' to 5', synthesize 5' to 3'
• The other strand (which has same sequence as RNA) is sometimes called the "coding" or "sense" strand, even though it is not read
• Some genes are on one strand; other genes are on the opposite strand

Example:

5' . . . ATGAATGTC . . . 3' sense

3' . . . TACTTACAT . . . 5' template

5' . . . augaauguc-> . . 3' RNA copy

RNA Polymerase Complex does all this:

recognizes and binds to promoter region

unwinds DNA

binds to RNA product

catalyzes phosphodiester bond formation

reanneals DNA

recognizes termination factor and releases DNA and RNA

Transcription in Prokaryotes

Initiation

• Special promoter sequences upstream of gene
• -35 TTGACA
• -10 TATAAT
• Promoters with different sequences bind less efficiently and have less transcription
• Sigma factor is needed for polymerase to recognize the promoter and to recognize the correct template strand.
• First base usually A or G
• First base has 5' ppp

Elongation:

• a "Transcription Bubble" migrates down the DNA
• ~17 bp of melted DNA
• ~8-12 bp of this paired with newly synthesized RNA
• ~1 turn of an 'A-type' helix
• • no more than this to prevent RNA from getting entwined in the DNA
• The RNA-DNA mini-helix can rotate about the DNA strand to constantly free the transcript
• • elongation moves at ~50 bases/sec.
  • No editing (unlike DNA synthesis)
• Accuracy: ~ 1 error in 100 bases
• • Much more error prone than DNA synthesis
  • Since mistakes are not passed on to daughter cells, they are not critical

See the figures in your book that show the transcription bubble. You should have a general sense of how the process works, although it is not necessary to memorize every part.

Termination

• Rho-independenet (hairpin)
• Rho-dependent

The critical point is that there are specific signals that cause RNA transcription to terminate.

We viewed an animation of RNA synthesis in class. You may want to go the the DNAi website to explore it some more.

Transcription in Eukaryotes

The general process is the same as in bacteria except :

• more than one RNA polymerase
• promoter and termination sequences are slightly different
• RNA processing (splicing, capping, adding poly A tail) is needed to make functional mRNA
• Eukaryotes have a nucleus, so RNA synthesis is separated from protein synthesis
• There are 3 RNA Polymerases
• • Pol I makes ribosomal RNAs
  • Pol II makes mRNA
  • • This is what produces most structural proteins
  • Pol III makes tRNAs and small nuclear RNAs

Initiation in Eukaryotes

Several transcription factors (TFII a,b,c, etc) recognize the promoters and combine with Polymerase II to start transcription (See Figure 5.6).

The critical point is not to memorize what each transcription factor does, but the know that there is some complex machinery that must be assembled before transcription can begin.

RNA Processing

• Capping: 5' end gets "capped" with a 7-methyl G
• Polyadenylation: 25 to 60 A's are added to 3' end of mRNA
• • Signal is AAUAAA
• Splicing: Splicosomes cut out introns, connect exons

None of that happens in prokaryotes.

See Fig. 5.9 for 5' cap and 5.10 for poly(A) tail formation.

Eukaryotic Genes have introns

• Introns are "intervening sequences" of DNA that are not present in mature RNA.
• Discovered in late 70s
• Generally thought to be non-functional (but recent evidence shows that there are some important sequence elements in introns).
• It is possible to see introns microscopically using DNA-RNA hybrids. In the hybrid molecule the introns show up as loops of single-stand DNA.
• • You might want to draw that to convince yourself

Alternative splicing

• Some genes have "alternative splicing", such that different exons are used in different tissues
• Example: troponin
• • Mature mRNA contains exons 1,2, and 4 in smooth muscle
  • Mature mRNA contains exons 1,3, and 4 in other tissues
• That may be an "easy" way for a single gene to have multiple functions