Chapter 14 Outline PDF

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Chapter 14 Outline...

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Sunday, February 19, 2017

Chapter 14 Outline RNA Molecules and RNA Processing - In eukaryotic cells, RNA molecules are often extensively modified after transcription: for genes that encode proteins, a special nucleotide called the cap is added to the 5’ end, a tail of adenine nucleotides is added to the 3’ end, and introns are cut out of the middle

Section 14.1 | Many Genes Have Complex Structures - In 1902, Garrod suggested tat genes encode proteins • Proteins are made of amino acids, so a gene contains the nucleotides that specify the amino acids of a protein - Gene Organization • Francis Crick in 1958 proposed that genes and proteins are collinear: that there is a direct correspondence between the nucleotide sequence of DNA and the amino acid sequence of a protein

• The concept of colinearity suggests that the number of nucleotides in a gene should be proportional to the number of amino acids in the protein encoded by the gene

- Concept: When a continuos sequence of nucleotides in DNA encodes a continuous sequence of amino acids in a protein, the two are said to be colinear. In eukaryotes, not all genes are colinear with the proteins that they encode

- Introns: • Many eukaryotic genes contain coding regions called exons and noncoding regions called intervening sequences or introns

• Introns are common in eukaryotic genes but are rare in bacterial genes - Introns are present in mitochondrial and chloroplast genes as well as the nuclear genes of eukaryotes • Geneticists have debated the evolutionary origin of introns - Intron late hypothesis: proposes that introns were absent from ancient organisms but were later acquired by eukaryotes

- Intron early hypothesis: suggests that early ancestors to bacteria, archaea, and eukaryotes possessed introns that were later lost by prokaryotes and simple eukaryotes

• Four major types of introns: - Group I: located in genes of eubacteria, bacteriophages, and eukaryotes; self-splicing - Group II: located in genes of eubacteria, archaea, and eukaryotic organelles; self-splicing - Nuclear pre-mRNA: located in protein-encoding genes in the nucleus of eukaryotes; spliceosomal - tRNA: located in tRNA genes of eubacteria, archaea, and eukaryotes; enzymatic - Concept: Many eukaryotic genes contain exons and introns. Both are transcribed into RNA but introns are later removed by RNA processing. The number and size of introns vary from gene to gene; they are common in many eukaryotic genes but uncommon in bacterial genes

- The Concept of the Gene Revisited • To define a gene as a sequence of nucleotides that encodes amino acids in a protein no longer seems appropriate because this definition excludes introns, which do not specify amino acids

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Sunday, February 19, 2017 - This definition also excludes nucleotides that encode the 5’ and 3’ ends of an mRNA molecule, which are required for translation but do not encode amino acids

• Many geneticists have broadened the concept of a gene to include all sequences in DNA that are transcribed into single RNA molecule

- Defined this way, a gene includes all exons, introns, and those sequences at the beginning and end of the RNA that are not translated into a protein

- Concept: The discovery of introns forced a reevaluation of the definition of the gene. Today, a gene is often defined as a DNA sequence that encodes an RNA molecule or the entire DNA sequence required to transcribe and encode an RNA molecule

Section 14.2 | Messenger RNAs, Which Encode the Amino Acid Sequences of Proteins, Are Modified after Transcription in Eukaryotes - As soon as DNA was identified as the source of genetic information, it became clear that DNA cannot directly encode proteins

• Alfred Hershey discovered a type of RNA that was synthesized rapidly after bacteriophage infection • These observations were consistent with the idea that RNA was copied from DNA and that this RNA then directed the synthesis of proteins

- At the time, ribosomes were known to be somehow implicated in protein synthesis, and much of the RNA in a cell was known to be in the form of ribosomes

• Ribosomes were believed to be the agents by which genetic information was moved to the cytoplasm for the production of protein

• Using equilibrium density gradient centrifugation, Brenner, Jacob, and Meselson demonstrated that it is not the case

- They showed that new ribosomes are not produced during the burst of protein synthesis that accompanies phage infection

- Francois Gros concluded that newly synthesized, short-lived RNA carries the genetic information for protein structure to the ribosome

• The term messenger RNA was coined - The Structure of Messenger RNA • Messenger RNA functions s the template for protein synthesis; it carries genetic information from DNA to a ribosome and helps to assemble amino acids in their correct order

• In the mRNA, each amino acid in a protein is specified by a set of three nucleotides called a codon - Both prokaryotic and eukaryotic mRNAs contain three primary regions - The 5’ untranslated region is a sequence of nucleotides at the 5’ end of the mRNA that does not encode any of the amino acids in a protein

- IN bacterial mRNA, this region contains a consensus sequence (UAAGGAGGU) called the Shine-Dalgarno sequence, which serves as the ribosome binding site during translation

• The next section of mRNA is the protein-coding region which comprises the codons that specify the amino acid sequence of the protein

- The protein-coding region begins with a start codon and ends with a stop codon

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Sunday, February 19, 2017 - The last region of mRNA is the 3’ unsaturated region, a sequence of nucleotides at the 3’ end of the mRNA and not translated into protein

- Concept: Messenger RNA molecules contain three major regions: a 5’ untranslated region, a protein-coding region, and a 3’ unsaturated region. The 5’ and 3’ untranslated regions do not encode any amino acids of a protein, but contain information that is important in translation, RNA stability and regulation of gene expression

- Pre-mRNA Processing • In bacterial cells, transcription and translation take place at the same time • Because transcription and translation are coupled, bacterial mRNA has little opportunity to be modified before protein synthesis

- The Addition of the 5’ Cap • Addition of 5’ cap: facilitates binding of ribosome to 5’ end of mRNA increases mRNA stability, enhances RNA splicing

• 3’ cleavage and addition of poly(A) tail: increases stability of mRNA, facilitates binding of ribosome to mRNA • RNA splicing: removes noncoding introns from pre-mRNA, facilitates export of mRNA to cytoplasm, allows for multiple proteins to be produced through alternative splicing

• RNA editing: alters nucleotide sequence of mRNA - Concepts: Eukaryotic pre-mRNAs are processed at their 5’ and 3’ ends. A cap, consisting of a modified nucleotide and several methyl groups is added to the 5’ end. The cap facilitates the binding of a ribosome, increases the stability of the mRNA, and may affect the removal of introns. Processing at the 3’ end includes cleavage downstream of an AAUAAA consensus sequence and the addition of a poly(A) tail

- Consensus Sequences and the Spliceosome • Splicing requires the presence of three sequences in the intron - One end of the intron is referred to as the 5’ splice site, and the other end is the 3’ splice site; these splice sites possess short consensus sequences

• The third sequence important for splicing is at the branch point, which is an adenine nucleotide that lies form 18 to 40 nucleotides upstream of the 3’ splice site

• Splicing takes place within a large structure called the spliceosome, which is one of the largest and most complex of all molecular structures

- Concept: Introns in nuclear genes contain three consensus sequences critical to splicing: a 5’ splice site, a 3’ splice site, and a branch complex called the spliceosome, which consists of snRNAs and proteins

- The Process of Splicing • Before splicing takes place, an intron lies between an upstream exon (exon 1) and a downstream (exon 2) • Pre-mRNA is spliced in two distinct steps - 1. The pre-mRNA is cut at the 5’ splice site • Lariat: looplike structure created in the splicing of nuclear pre-mRNA in which the 5’ end of an intron is attached to a branch point in pre-mRNA

- 2. A cut is made at the 3’ splice site and the 3’ splice site and at the same time the 3’ end of sexton 1 becomes covalently attached (spliced) to the 5’ end of exon 2

• These splicing reactions take place within the spliceosome, which assembles on the pre-mRNA in a step-bystep fashion and carries out the splicing reactions

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Sunday, February 19, 2017 - A crucial feature of the process is a series of interactions between the mRNA and the snRNAs and between different snRNAs

• Most mRNAs are produced from single pre-mRNA molecule with the exons are spliced together - Trans-splicing: the process of splicing exons from two or more pre-mRNAs • RNA splicing, which takes place in the nucleus, must be done before the RNA can move into the cytoplasm - Concept: Intron splicing of nuclear genes is two-step process: (1) the 5’ end of the intron is cleaved and attached to the branch point to form a lariat and (2) the 3’ end of the intron is cleaved and the ends of the two exons are spliced together. In the process the exons are joined and the intervening intron is removed. These reactions take place within the spliceosome

- Minor Splicing • Some introns in the pre-mRNAs of multicellular eukaryotes utilize a different process of intron removal known as minor splicing

- Self-Splicing Introns • Some introns are self-splicing: they possess the ability to remove themselves from an RNA molecule • Group I introns are found in a variety of genes, including some rRNA genes in protests, some mitochondrial genes in fungi, and even some bacterial and bacteriophage genes

• Group II introns, presents in genes of eubacteria, archaea, and eukaryotic organelles, also have the ability to self-splice

- All group II introns also fold into secondary structures - Concept: Some introns are removed by the minor splicing system. Other introns are self-splicing and consist of two types: group I introns and group II introns. These introns have complex secondary structures that enable them to catalyze their excision from RNA molecules without the aid of enzymes or other proteins

- Alternative Processing Pathways • A finding that complicates the view of a gene as a sequence of nucleotides that specifies the amino acid sequence of protein is the existence of alternative processing pathways

- In these pathways, a single pre-mRNA is processed in different ways to produce alternative types of mRNA, resulting in the production different proteins from the same DNA sequence

• One type of alternative processing is alternative splicing, in which the same pre-mRNA can be spliced in more than one way to yield multiple mRNAs that are translated into different amino acid sequences and thus different proteins

- Another type of alternative processing requires the use of multiple 3’ cleavage sites, where two or more potential sites for cleavage and polyadenylation are present in the pre-mRNA

- Both alternative splicing and multiple 3’ cleavage sites can exist in the same pre-mRNA transcript • Alternative processing of pre-mRNAs is common in multicellular eukaryotes - Alternative splicing may play a role in organism complexity - Concept: Alternative splicing enables exons to be spliced together in different combinations to yield mRNAs that encode different proteins. Alternative 3’ cleavage sites allow pre-mRNA to be cleaved at different sites

- RNA Editing • The assumption that all information about the amino acid sequence of protein resides in DNA is violated by a process called RNA editing

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Sunday, February 19, 2017 - In RNA editing, the coding sequence of an mRNA molecule is altered after transcription, so the protein has an amino acid sequence that differs from that encoded by the gene

• A variety of mechanisms can bring about changes in RNA sequences - In some cases, molecules called guide RNAs play a crucial role - A gRNA contains sequences that are partly complementary to segments of the predated RNA and the two molecules undergo base pairing in these sequences

- Concept: Individual nucleotides in the interior of pre-mRNA may be changed, added, or deleted by RNA editing. The amino acid sequence produced by the edited mRNA is not the same as that encoded by RNA

Section 14.3 | Transfer RNAs, Which Attach to Amino Acids, Are Modified after Transcription in Bacterial and Eukaryotic Cells - In 1956, Francis Crick proposed the idea of a molecule that transports amino acids to the ribosome and interacts with codons in mRNA, placing amino acids in their proper order in protein synthesis

- Each tRNA is capable of attaching to only one type of amino acid • The complex of tRNA plus its amino acid can be written in abbreviated form by adding a three-letter superscript representing the amino acid to the term tRNA

- The Structure of Transfer RNA • A unique feature of tRNA is the occurrence of rare modified bases • All RNAs have the four standard bases (adenine, cytosine, guanine, and uracil) specified by DNA, but tRNAs have additional bases including ribothymine, pseudouridine and dozens of others

• Modified bases arise form chemical changes made to the four standard bases after transcription - These changes are carried out by special tRNA-modifying enzymes • The structures of all tRNAs are similar - Cloverleaf structure: secondary structure common to all tRNAs • The TψC arm is named for the bases of three nucleotides in the loop of this arm: thymine (T), pseudouridine (ψ), and cytosine (C)

- The anticodon arm lies at the bottom of the tRNA - The nucleotides at the end of this arm make up the anticodon, which pairs with the corresponding codon on mRNA to ensure that the amino acids link in the correct order

• Although each tRNA molecule folds into a cloverleaf owing to the complementary pairing of bases, the cloverleaf is not the 3D (tertiary) structure of tRNAs found in the cell

- Transfer RNA Gene Structure and Processing • The genes that produce tRNAs may be in clusters or scattered about the genome • There is no generic processing pathway for all tRNAs: different tRNAs are processed in different ways • Some eukaryotic and archaea tRNA genes possess introns of variable length that must be removed in processing

- Concept: All tRNAs are similar in size and have a common secondary structure known as the cloverleaf. Transfer RNAs contain modified bases and are extensively processed after transcription in both bacterial and eukaryotic cells

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Sunday, February 19, 2017 Section 14.4 | Ribsomal RNA, a Component of the Ribosome, Is Also Processed After Transcription - Within ribosome, the genetic instructions contain in mRNA are translated into the amino acid sequences of polypeptides

• Ribosomes play an integral part in the transfer of genetic information from genotype to phenotype - The Structure of the Ribosome • The ribosome is one of the most abundant molecular complex in the cell: a single bacterial cell may contain as many as 20,000 ribosomes, and eukaryotic cells possess more

• A functional ribosome consists of two subunits, a large ribosomal subunit and a small ribosomal subunit, each of which consists of one or more pieces of RNA and a number of proteins

- Ribosomal RNA Gene Structure and Processing • The genes for rRNA can be present in multiple copies and the numbers vary among species: all copies of the rRNA gene in a species are identical or nearly identical

• Eukaryotic cells possess two types of rRNA genes: a large gene that encodes 18S rRNA, 28S rRNA, and 5.8S rRNA, and a small gene that encodes the 5S rRNA

• The processing of rRNA and ribosome assembly in eukaryotes take place in the nucleolus - Concept: A ribosome is a complex organelle consisting of several rRNA molecules and many proteins. Each functional ribosome consists of a large and small subunit. Ribosomal RNAs in both bacterial and eukaryotic cells are modified after transcription. In eukaryotes, rRNA processing is carried out by small nucleolar RNAs

Section 14.5 | Small RNA Molecules Participate in a Variety of Functions - Must evidence suggests that the first genetic material was RNA and early life was dominated by RNA molecules - RNA Interference • Fire, Mello, and their colleagues observed something strange: they were inhibiting the expressions of genes in a nematode by inserting single-stranded RNA molecules that were complementary to a gene’s DNA sequence

• These initial studies led to the discovery of small RNA molecules that are important in gene silencing • Subsequent research revealed a large array of small RNA molecules with important cellular functions in eukaryotes, which now include at least three major classes: small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs), depending on their origin and mode of function

• RNA interference (RNAi) is a powerful and precise mechanism used by eukaryotic cells to limit the invasion of foreign genes and to censor the expression of their own genes

- Small Interfering and Micro RNAs • Two abundant classes of RNA molecules that function in RNA interference in eukaryotes are small interfering RNAs and microRNAs

• siRNA: - Origin: mRNA, transposon, virus - Cleavage of: RNA duplex or single-stranded RNA that forms long hairpins - Size: 21-25 nucleotides - Action: degradation of mRNA, inhibition of transcription, chromatin modification

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Sunday, February 19, 2017 - Target: genes from which they were transcribed • miRNA: - Origin: RNA transcribed from distinct gene - Cleavage of: Single-stranded RNA that forms short hairpins of double-stranded RNA - Size: 21-25 nucleotides - Action: degradation of mRNA, inhibition of translation, chromatin modification - Target: genes other than those form which they were transcribed • Both siRNA and miRNA molecules combine with proteins to form an RNA-induced silencing complex - Key to the functioning of RISCs is a protein called Argonaute - Processing and Function of miRNAs and siRNAs • The genes that encode miRNAs are transcribed into longer precursors, called primary miRNA that range from several hundred to several thousand nucleotides in length

• The pri-miRNA is then cleaved into one or more smaller RNA molecules with a hairpin • The RISC attaches to a complementary sequence on the mRNA, usually in the 3’ untranslated region of the mRNA

- Concept: Small interfering RNAs and microRNAs are tiny RNAs produce when large, double-stranded RNA molecules are cleaved by the enzyme Dicer. Small interfering RNAs and microRNAs participate in a variety of processes, including mRNA degradation, the inhibition of translation, the methylation of DNA, and chromatin remodeling

- Piwi-Interacting RNAs • Piwi-interacting RNAs (piRNA) were discovered in 2001 - They are somewhat longer than siRNAs and miRNAs, consisting of 24 to 30 nucleotides and are derived from long single-stranded RNA transcripts

• Piwi interacting RNAs combine with Piwi proteins and suppress the expression and movement of transposes in the germ cells of animals

• CRISPR RNA - CRISPR RNAs (crRNAs) were discovered in prokaryotes: crRNAs are encoded by DNA sequences found in bacterial and archaea ge...