Lecture 23

• overview of transcription

  synthesis of RNA from a DNA template is carried out by RNA pol

  mechanism is same in prokaryotes and eukaryotes, but processing of RNA product is different

  takes place in three stages: initiation, elongation, termination
• DNA strands

  Most DNA is ds, and in a given region of DNA, only one of the two strands is copied during RNA synthesis. This is different from replication of DNA in which both strands are copied.
• coding strand

  has the same directionality and sequence as the product RNA, except that it contains T residues, whereas the product RNA contains U residues. The coding strand does not serve as a template for RNA synthesis.
• template/ antisense strand

  serves as a template for transcription. The DNA template is complementary and antiparallel to the product RNA.
• DNA vs mRNA

  DNA contains 2-deoxyribose in place of ribose. The template strand is antiparallel and complementary to both the coding strand and RNA product.
• template strand of DNA

  provides the information for messenger RNA synthesis.
• mRNA and tRNA

  act as intermediaries between DNA and protein
• functions of RNA pol part 2

  detects termination signals that specify where a transcript ends

  interacts with activator and repressor proteins that modulate the rate of transcription initiation over a wide range
• initiation of transcription

  initiated at promoter sites on the DNA template:

  • Prokaryotic promoters are generally centered at about 10 and 35 nucleotides upstream of the start site
• consensus sequence

  deduced from analyses of many prokaryotic promoters, are TTGACA (-35) and TATAAT (-10)

  distance between the consensus sequences: separation of 17 nucleotides is optimal
• promotors in transcription

  Promoters differ markedly in their efficacy. Genes with strong promoters are transcribed every 2 seconds (E. coli)

  genes with very weak promoters are transcribed about once every 10 minutes
• prokaryotic promotors

  promoters consist of 2 components (10 and 35 nucleotides upstream) The most common consensus sequence for prokaryotic organisms, deduced from analyses of many promoters, is TTGACA (-35) and TATAAT (-10). The -10 sequence is sometimes referred to as a Pribnow Box
• RNA polymerase structure

  consists of 5 subunits

  four of them are alpha 2 beta beta'

  sigma subunit is a transient component of the complex (recognizes promotor sites, can not start transcription without)
• functions of sigma subunits

  decreases RNA polymerase’s affinity for general regions of DNA by a factor of 10^4

  enables RNA polymerase to recognize promoter sites
• holoenzyme

  slides along the DNA until it encounters a promoter region

  rate constant for binding of RNA polymerase to the promoter is 100 times faster by sliding than could be accomplished by repeatedly moving on and off the DNA.
• DNA unwinding

  Prior to transcription of an area of DNA, the RNA polymerase unwinds about 17 bp of template DNA to produce an open "transcription bubble". The DNA is rewound at the rear end of the polymerase as transcription of an area is completed.
• 5' triphosphate tag

  Transcription is initiated without a primer; the usual first residue is a 5'-GTP or ATP. The first residue transcribed retains a 5' triphosphate "tag".
• transcription bubble

  transcription in progress in an open transcription bubble with a growing chain of nascent RNA hybridized to the template strand of DNA. There is a triphosphate residue at the 5’ end of the RNA chain that will be retained in the final RNA product
• Only the template strand is transcribed into RNA. The coding strand is not used for this purpose. (However, the same strand does not always serve as a template. A DNA strand that functions as a template for one gene can be the coding strand for other genes.)
• RNA synthesis can be initiated at specific locations without a primer. This contrasts to DNA synthesis, which requires a primer for initiation
• no permanent separation of the two DNA strands during transcription, occurs in an open bubble of ss DNA (17 nt in length)

  After transcription, two strands of DNA hybridize back to ds
• rna polymerase reaction requirements

  A DNA template (ds or ss)

  four ribonucleoside triphosphates (ATP, GTP, UTP, and CTP)

  DNA-dependent RNA polymerase

  variety of ancillary proteins

  divalent metal ion such as Mg2+

  does not require a primer
• RNA polymerase step 1

  new RNA is added onto 3' end of growing RNA strand (5' to 3')

  free 3' OH group on C residue, empty binding site for UTP across from A residue
• rna polymerase step 2

  UTP will diffuse to occupy open site

  U residue can H bond with A (AU bp)

  3' OH on C with attack the P atom of alpha P UTP
• rna polymerase step 3

  3' OH of C forms a phosphodiester linkage with alpha-P of UTP, releases inorganic pyrophosphate, hydrolyzed to 2Pi

  growing RNA strand one nt longer

  free 3'OH for ATP across from a T residue
• rna polymerase step 4

  ATP will diffuse to occupy open site

  a with H bond to T

  3' OH of U residue will NU attack on P atom of the alpha-P of ATP
• rna polymerase 5

  formation of phosphodiester linkage between 3'OH and alpha-P of ATP, releases inorganic pyrophosphate

  growing RNA strand

  same pattern of elongation will occurs
• In prokaryotes, transcription and translation both take place in the cytoplasm. In eukaryotes, transcription and processing take place in the nucleus, and translation takes place in the cytoplasm
• In prokaryotes, there is one RNA polymerase that does it all. In eukaryotes, there are three RNA polymerases with divergent functions.
• In prokaryotes, there is little processing of mRNA. Eukaryotic RNA is processed by capping at the 5'-end, addition of a poly-A tail, and removal of introns (splicing of exons).
• RNA polymerase 1

  RNA polymerase I is located in nucleoli, and transcribes the tandem array of genes for 18S, 5.8S, and 28S ribosomal RNA
• rna polymerase 3

  The other ribosomal RNA molecule (5S rRNA) and all the transfer RNA molecules are synthesized by RNA polymerase III, which is located in the nucleoplasm rather than in the nucleoli
• rna polymerase 2

  which is located in the nucleoplasm, synthesizes the precursors of messenger RNA as well as several small RNA molecules, such as those of the splicing apparatus
• The nucleus of eukaryotes contains three types of RNA polymerase that differ in template specificity, location within the nucleus, and susceptibility to inhibitors
• RNA processing in eukaryotes

  capping of the 5’end of the RNA strand, the addition of a poly-A tail, and the removal of introns followed by the joining of exons
• capping of the 5' end of RNA

  Before RNA is exported from nucleus, the 5’ end of the molecule is modified

  Modifications: addition of a 7- methylguanylate residue attached by a triphosphate linkage to the terminal ribose group

  last two ribose residues are sometimes methylated
• addition of a 3' poly A tail

  The 3’ end of the messenger RNA is also modified. At that end, a specific endonuclease cleaves the mRNA downstream of an AAUAAA sequence, and then a poly(A) polymerase adds about 250 adenylate residues
• processing of introns

  DNA within the gene that do no code for amino acids in the final protein product

  During transcription of genes that contain introns, the entire gene is transcribed into mRNA, the primary transcript is then modified with a cap and poly(A) tail, and then the introns are removed
• splicing of exons

  When introns are removed, the two adjoining exon segments of the RNA must be spliced together

  There are consensus sequences of GU at the 5' end of the intron, and a pyrimidine rich region followed by AG at the 3' end, that delineate the points at which splicing occurs
• mechanism of splicing

  The upstream (first) exon is attacked at its 3'-end by the 2' OH group of a specific A residue within the first intron. (The branch-site adenosine forms a lariat intermediate with the 5' ribose of the first intron.)