EFB325 Cell Physiology

Transcription

According to the central dogma, the sequence in DNA is used to produce RNA, which is then translated into a sequence of amino acids in a polypeptide

• the rate of initiation of transcription is the key determinant of how much RNA is made and consequently, how much protein is made from a particular gene
• only limited regions of a chromosome represent genes=region of DNA that encodes a specific function(s) and the basic unit of heredity - genes code for proteins or functional RNAs (like rRNA and tRNA)
• those regions of DNA are first transcribed into RNA by RNA polymerase
• RNA is nearly always single-stranded, although tRNAs have regions which are double-stranded
• RNA polymerase uses one of the DNA strands as a template, synthesizing an RNA polymer using ribonucleoside triphosphates as building blocks (the other strand, not used in transcription, is called the coding strand, because it has the same sequence as the RNA-except Ts in DNA are Us in RNA)
• the appropriate new ribonucleotide to add is determined by proper base pairing with the template to produce a complementary new strand (Remember: U pairs with A)
• RNA polymerase adds new ribonucleotides to the 3' end of the new RNA polymer, so synthesis proceeds from 5'->3' - referring to the new strand (as with DNA polymerase)
• the building blocks of RNA (substrate for RNA polymerase) are: ATP, CTP, GTP, and UTP
• the RNA molecules that are synthesized are transient intermediates = they are degraded relatively quickly after they are synthesized (as soon as a few minutes to as long as 12-24 hours) - rRNA tends to be more stable than mRNA

Rate of cellular metabolism, responses to stimuli, and cellular differentiation depend on differences in the types of proteins present in a cell and their relative levels of accumulation & activity

• the presence of a protein in a particular cell and how much active protein accumulates can be regulated in many different ways = regulation of gene expression
• the amount of active protein that is synthesized largely depends on the amount of mRNA present that encodes that protein
• therefore: transcription initiation is the primary way that cells control which proteins are made in a particular cell, when they are made, and how much is made

There are fundamental differences between prokaryotes and eukaryotes in some of the specific mechanisms of transcription and translation. Notice these differences and think about why the eukaryotic systems tend to be much more complex.

There is 1 type of RNA polymerase in prokaryotes and 3 types of RNA polymerase in eukaryotes

• in prokaryotes, all genes are transcribed by the same (the only type) of RNA polymerase

In eukaryotes:

• the large rRNAs are transcribed by RNA polymerase I in the nucleolus
• the mRNAs, which are used in translation to make proteins, are transcribed by RNA polymerase II
• the smallest rRNA and tRNAs are transcribed by RNA polymerase III
• RNA polymerase II is strongly inhibited by alpha-amanitin, a toxin produced by Amanita mushrooms (death caps)

Transcription in prokaryotes starts at the promoter and ends at the terminator

• RNA polymerase binds to a specific region of DNA=promoter, which is recognized by a subunit of the RNA polymerase=sigma factor
• the DNA sequence of the promoter is fairly short, and promoters from different genes have very similar DNA sequences
• NOTE: prokaryotic RNA polymerase does not require any other proteins in order to initiate transcription
• RNA polymerase unwinds a segment of the double-stranded DNA to expose the template strand
• the first ribonucleoside triphosphate base-pairs with a specific base on the template (start point), then a second ribonucleoside triphosphate base-pairs to the next position on the template
• RNA polymerase joins the two, then proceeds to add bases to the 3' end, according to the proper base-pairing with the template strand
• after several bases have been added, then the sigma factor dissociates
• RNA polymerase continues to unwind the DNA, build the RNA strand, then rewind the DNA as it proceeds (about 18 base pairs are unwound where the RNA polymerase is actively transcribing)
• when the RNA polymerase reaches the terminator region, the terminator signals the RNA polymerase to dissociate from the DNA and release the completed RNA transcript
• at this point in prokaryotes, the RNA is ready for translation

Note:

• RNA polymerase does not need a primer, like DNA polymerase
• RNA polymerase does not have a proofreading activity
• both RNA and DNA polymerase add new bases from 5'->3', using triphosphates as building blocks
• both RNA and DNA polymerase use single-stranded DNA as a template for producing a new strand with complementary sequence

The different RNA polymerases in eukaryotes bind to different types of promoters

• but in general, promoters are the site of initiation of transcription and have particular DNA sequences involved in the binding of RNA polymerase
• specificity is conferred by protein interactions with particular sequences in the DNA

In eukaryotes, RNA polymerase II transcribes genes to produce mRNAs encoding proteins

• the core promoter is the minimal region of the DNA needed to initiate transcription by RNA polymerase II
• the core promoter has a short region of DNA=TATA box, with a sequence that is nearly identical in all genes transcribed by RNA polymerase II
• transcription is initiated when the TATA-binding protein binds to the TATA box region of DNA, then other transcription factors (proteins) bind to the promoter and to each other
• next RNA polymerase II binds to the promoter/transcription factor complex and unwinds the DNA
• synthesis of the RNA strand begins at the start site and proceeds, with the addition of bases to the 3' end of the new RNA strand, which is complementary to the template DNA
• it is not known what signals RNA polymerase II to stop and dissociate from the DNA
• NOTE: eukaryotic RNA polymerase requires other proteins (the transcription factors) in order to bind to the promoter and initiate transcription

In eukaryotes, there is further processing of the RNA transcript in the nucleus before protein synthesis can occur (in the cytoplasm)

• most eukaryotic mRNAs are modified in the nucleus before they are exported to the cytoplasm to be translated into proteins

5' cap

• all mRNAs have a modified guanosine residue (7-methyl guanosine) added to the 5' end=5' cap
• this base is linked to the 5' end by a different bonding arrangement-a 5'-5' triphosphate bridge
• the 5' cap protects the RNA from being degraded by ribonucleases from the 5' end and also positions the mRNA on the ribosome during initiation of translation

3' cleavage, then polyadenylation (addition of a poly(A) tail)

• most eukaryotic mRNAs are cleaved at the 3' end at a site next to a region with a specific RNA sequence
• after cleavage, an enzyme adds a long string of A's onto the 3' end=poly(A) tail
• the poly(A) tail also helps protect the RNA from being degraded by ribonucleases from the 3' end and also aids in transport to the cytoplasm and in binding to ribosomes

intron excision=RNA splicing

• most eukaryotic genes include specific regions that are transcribed, but that do not contain sequence meant to encode protein, so those regions are removed from the RNA=introns (intervening sequence)
• the regions which remain and encode protein sequence are exons (expressed sequence)
• the process of removing the introns is called RNA splicing and for most genes is coordinated by a complex composed of RNA and proteins that bind to the RNA in the regions of the introns=spliceosome
• these RNA-protein complexes are called snRNPs (snurps, small nuclear ribonucleoproteins)
• splicing involves cleaving the RNA at the 5' end of the intron (the 5' end of the intron loops back and links with a base in the middle of the intron RNA=branchpoint), then the free 3' end of exon 1 links with the 5' end of exon 2 and the bond with the 3' end of the intron is broken, releasing the piece of intron RNA (which is in a loop called a lariat)
• splicing of some genes does not require snRNPs (the introns are self-splicing), the splicing reaction is catalyzed by the RNA itself=ribozyme

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