Eukaryotic Module 11

evidence that DNA is the genetic material

• in 1908, Archibald Garrod found that people with alcaptonuria lacked an enzyme in their blood that oxidized homogentisic acid, a compound formed during the breakdown of Phe and Tyr

• in the 1940s, George Beadle and Edward Tatum isolated dozens of mutants in Neurospora and proposed the “one gene-one enzyme” hypothesis

• in 1956, Vernon Ingram reported that a substitution of a valine for a Glu causes sickle cell anemia

the beadle-tatum experiment

• isolation of genetic mutants in neurospora

• helped form the idea that genes direct the production of proteins

• neurospora grows in a simple medium containing a single organic carbon source (like a sugar), inorganic salts, and biotin

  • so it was thought that it synthesized all its required metabolites

• the idea was that an organism with such broad synthetic capacity should be very sensitive to enzymatic deficiencies, which would then be detected using the proper experimental protocol

• they used to radiation to damage the DNA in the spores

• they screened for mutations affecting enzymes by testing their capability to grow in a minimal medium that lacked the essential compounds known to be synthesized by this organism

• if a spore was unable to grow in the minimal medium, but a genetically identical sore can grow in a medium supplemented with a particular metabolic compound, then the researchers could conclude the cells have an enzymatic deficiency that prevents them from synthesizing this essential compound

• out of over a thousand cells, two of the spores were unable to grow on the minimal medium; one needed pyridoxine (vitamin B6) and the other needed thiamine (B1)

• out of dozens of mutants isolated, each had a gene defect resulting in an enzyme deficiency that prevented the cells from catalyzing a particular metabolic reaction

• this led to the conclusion that a gene carries the information for the construction of a particular enzyme (one gene-one enzyme hypothesis)

• later become the one gene-one polypeptide hypothesis once it was learned that enzymes are often composed of more than one polypeptide, each encoded by its own gene

• this was also modified when it was found that a single gene often generates a variety of polypeptides as a result of alternative splicing

picture of beadle-tatum experiment

flow of information in eukaryotes

• transcription: synthesis of an RNA from a DNA template

• translation: synthesis of a protein from a mRNA molecule

RNAs

• mRNAs: RNAs that include nucleotide code information for polypeptide synthesis

• rRNAs: RNA components of ribosomes

• tRNAs: RNAs that translate the information in the mRNA nucleotide code into the amino acid order of a polypeptide

• other RNAs: small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs), microRNAs (miRNAs)

structure of RNAs

• many fold into a complex 3D shape, which is different from one type of RNA to another

• RNA folding is driven by the formation of regions of having complementary pairs

• RNAs often contain nonstandard base pairs and modified nitrogenous bases

• the unorthodox regions serve as recognition sites for proteins and other RNAs, promote RNA folding, and help stabilize the structure of the molecule

transcription

• DNA-dependent RNA polymerases (RNA polymerases): the enzymes responsible for transcription in both prokaryotic and eukaryotic cells

  • incorporate nucleotides into a strand of RNA with a sequence complementary to the template DNA strand

• template: the DNA strand whose sequence is complementary to the RNA molecule synthesized from it

• promoter: the site on DNA to which an RNA polymerase molecule binds prior to initiating transcription

• transcription factors: the proteins that are required to help RNA polymerases to recognize promoters

  • also contains info that determines which DNA strand is transcribed and the site it begins at

transcription direction and energetics

• catalytic rate is about 20-50 nt/s

• chain elongation: attack by the 3’ OH of the nucleotide at the growing strand, end on the 5’ P of the incoming nucleoside triphosphate

• RNA pols move along the template strand 3’→5’ direction

• as the pol progresses along, the template strand is unwound and the pol assembles a complementary strand of RNA that grows 5’→3’

  • overwound DNA ahead of the pol and underwound behind

• favorable reactions

transcription in prokaryotes

• a single type of RNA pol composed of 5 subunits that are tightly associated to form a core enzyme

• an accessory polypeptide, sigma factors (s), increases the enzyme’s affinity for promoter sites in DNA

• the polymerase, s factor, and DNA with the strands separated is called the open complex

• once about 10 nucleotides have been incorporated, the enzyme undergoes a conformational change into a transcriptional elongation complex that can move processively

the basic elements of a promoter region in the DNA of E. coli

• +1 nucleotide: where transcription is initiated

• -1 nucleotide: preceding the +1 nucleotide

• upstream: the portions of DNA preceding the initiation site

• downstream: the portions of DNA succeeding the initiation site

• consensus sequence: present in the promoters of many genes

• -35 element: the TTGACA consensus sequence around -35 site

• pribnow box (-10 element): the TATAAT consensus sequence around -10 site

initiation and termination of transcription in prokaryotes

• bacterial cells possess a variety of sigma factors that recognize different versions of the promoter sequence

• sigma 70 is a “housekeeping” sigma factor that initiates transcription of most genes

• alternative sigma factors initiate transcription of a small number of specific genes that participate in a common response

• bacterial transcription terminates when a specific nucleotide sequence is reached

• in about half of the cases, a ring-shaped rho protein is required for terminatioon

• in other cases, the polymerase stops transcription when it reaches a terminator sequence and releases the completed RNA chain

RNA polymerases in eukaryotes

• RNA pol I: synthesizes larger ribosomal RNAs (28S, 18S, and 5.8S)

• RNA pol II: synthesizes mRNA and most small nuclear RNAs (snRNAs and snoRNAs)

• RNA pol III: synthesizes various low MW RNAs, like tRNAs, 5S rRNA, and U6 snRNA

transcription and RNA processing in eukaryotes

• transcription requires a large variety of accessory proteins called transcription factors

• RNAs are transcribed as a longer precursor RNA transcript or pre-RNA; the corresponding segment of DNA is a transcription unit

• the primary RNA transcript is processed into smaller, functional RNAs

• plants have two additional RNA pols (IV and V) that are not essential for life and synthesize siRNAs

importance of rRNA

• more than 80% of the RNA in most cells consist of rRNA

• these rRNAs and many ribosomal proteins form ribosomes

rDNA

• the DNA sequences that encode rRNA

• normally repeated hundreds of times and are clustered in one or a few region of the genome

• clusters of rDNA are gathered together as part of one or more irregular shaped nuclear structures called nucleoli (nucleolus is plural)

the nucleoli

• produces ribosomes

• most of a nucleolus is composed of ribosomal subunits that give the nucleolus a granular appearance

  • fibrillar center (fc): contains rDNA

  • dense fibrillar component (dfc): contains the nascent pre-rRNA transcripts and associated proteins

  • granular component (gc): contains ribosomal subunits in various stages of assembly

four rRNAs of eukaryotes

• eukaryotes have 4 distinct rRNAs

• three in the large subunit and one in the small subunit

  • 28S, 5.8S, and 5S in large, and 18S in small

synthesis and processing of the rRNA precursor

• the 28S, 18S, and 5.8S are carved by various nucleases from a single primary transcript, the pre-rRNA

  • pre-rRNA is synthesized by RNA pol I

• 5S rRNA is synthesized from a separate RNA precursor outside of the nucleolus by RNA pol III

• the pre-rRNA has a large number of methylated nucleotides and pseudouridine residues

• when the precursor is first cleaved (in humans), over 100 methyl groups have been added to ribose groups in the molecule and approx. 95 of its uridine residues have been enzymatically converted to pseudouridine

  • all of these modifications occur post-transcriptionally

  • purpose of modifications could be to protect from cleavage, promote folding, or promote interactions with other molecules

• all of the nucleotides that are altered remain as part of the final products, while unaltered sections are discarded during processing

role of snoRNAs in the pre-rRNA processing

• a large number of snoRNAs are packaged with particular proteins to form particles (snoRNPs, small nucleolar ribonucleoproteins), which begin to associate with the rRNA precursors before it is fully transcribed

• snoRNPs function in processing of pre-rRNAs, such as removal of the 5’ end of the transcript and cleavage of the pre-RNAs

• some snoRNAs are present in low abundance, and are divided into two groups

  • one group determines which nucleotides on the pre-rRNA will have methylated ribose

  • the other group determines which uridines are converted to pseudouridines

synthesis and processing of the 5S rRNA

• about 120 nucleotides

• in eukaryotes, the 5S RNA molecules are encoded by a large number of identical genes that are separate from other rRNA genes and located outside the nucleolus

  • genes are also organized in a tandem array, and alternating with nontranscribed spacers

  • the promoter sequence lies within the coding section of the gene

• 5S RNA genes are transcribed by RNA pol III, which can bind to a promoter site located within the transcribed portion of the target gene

• the 5’ end of the primary transcript (pre-rRNA) is identical with that of the mature 5S RNA, but the 3’ end usually contains extra nucleotides that are removed during processing

• after synthesis, the 5S RNA is transported to the nucleolus to join the other components involved in the assembly of ribosomal subunits

synthesis and processing of the tRNAs

• there are about 50 different species of tRNA, each encoded by a repeated DNA sequence

• tRNA genes are found in small clusters scattered around the genome

  • a single cluster contains multiple multiple copies of different tRNA genes

• the DNA sequence encoding a tRNA is found in more than one cluster

• in a cluster, tRNA genes are separated by non-transcribed spacers

  • the promoter sequence lies within the coding section of the gene

• transcribed by RNA pol III

• the primary transcript of a tRNA is larger than the final product, and pieces of both the 5’ and 3’ sides of the pre-tRNA are trimmed

  • sometimes a small interior piece is trimmed

• all mature tRNAs have the triplet sequence CCA at their 3’ end; encoded in the tRNA gene in prokaryotes and added enzymatically after the tRNA is processed in eukaryotes

synthesis of mRNA

• all eukaryotic mRNA precursors are synthesized by RNA pol II, which is composed of 12 different conserved subunits

• many general transcription factors (GTFs) are required to initiate transcription along with RNA pol II

• promoters for RNA pol II lie to the 5’ end of each transcription unit

• a TATA box lies between 24 and 32 bases from the transcription start site

  • site of assembly of a pre-initiation complex (PIC) that contains the GTFs and RNA pol II

formation of the PIC

• the PIC assembly starts with the binding of the TATA-binding protein (TBP) to the promoter

  • TBP is a subunit of the TFIID (a GTF)

• TBP binding to the promoter causes a conformational change in DNA

• TFIID binding enables the assembly of a complete PIC

• the 3 GTFs bound to the promoter allows the binding of RNA pol II with its TFIIF

• all of the GTFs together at the promoter region is referred to as the PIC

steps of PIC assembly

• TFIID binds

• TFIIA and TFIIB come in; A helps TBP and B bridges the gap

• TFIIF comes in and then recruits RNA pol II

• TFIIE and TFIIH come in; E melts DNA and H acts as a helicase

mRNA transcription

• binding of the TBP to an RNA pol II promoter causes a dramatic distortion of the DNA

• TBP inserts itself into the minor groove of the double helix, bending the DNA molecule more than 80 degrees

• while TBP binds TATA, other subunits of the TFIID complex bind to other regions of the DNA

• as long as TFIID remains bound to the promoter, additional RNA pols may be able to attach for additional rounds of transcription

specific transcription factors

• a variety of specific TFs are required to bind at numerous sites in the regulatory regions of the promoter DNA to determine whether or not a PIC assembles at a promoter, and the rate the pol initiates new rounds of transcription from that promoter

termination of transcription by RNA pol II

• not well understood

• a transcribing RNA pol II can travel a variable/extensive distance past the point that will ultimately give rise to the 3’ terminus of the transcribed mRNA

heterogeneous nuclear RNAs (hnRNAs)

• RNAs of diverse (heterogeneous) nucleotide sequence

• only in the nucleus

• have large MWs (up to 80S, and 50,000 nt)

pulse chase results for various RNAs

• pulse for 30 min and chase 3 hours

• most of the RNA in the cell is present as 18S and 28S rRNA (and small RNAs)

• neither hnRNA or mRNA constitute a significant fraction of the RNA in the cell

• mRNA and its precursor hnRNA constitute a large percentage of the RNA that is being synthesized by the cell at any given time

• hnRNAs and mRNA are degraded after brief periods of time

  • half-lives of mRNA range from about 15 min to days

• tRNAs and rRNAs have half-lives ranging from days to weeks

properties of mRNAs

• contain a continuous sequence of nucleotides encoding a specific polypeptide

• found in the cytoplasm

• attached to ribosomes when they are translated

• most mRNAs contain 5’ and 3’ untranslated regions (UTRs), which have important regulatory functions

• eukaryotic mRNAs have special modifications at their 5’ and 3’ terminals

  • 3’ end has a string of 50-250 adenosine residues that form a poly(A) tail

  • 5’ end has a methylated guanosine cap